MODIFIED RNAi AGENTS

ABSTRACT

One aspect of the present invention relates to double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene. The dsRNA duplex comprises one or more motifs of three identical modifications on three consecutive nucleotides in one or both strand, particularly at or near the cleavage site of the strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 16/165,343, filed Oct. 19, 2018, which is a continuation of U.S. patent application Ser. No. 15/706,389, filed Sep. 15, 2017, which is a continuation of U.S. patent application Ser. No. 14/358,009, filed May 13, 2014, now U.S. Pat. No. 9,796,974, which claims priority to PCT Application No. PCT/US2012/065601, filed Nov. 16, 2012, which claims benefit of priority to U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011; all of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to RNAi duplex agents having particular motifs that are advantageous for inhibition of target gene expression, as well as RNAi compositions suitable for therapeutic use. Additionally, the invention provides methods of inhibiting the expression of a target gene by administering these RNAi duplex agents, e.g., for the treatment of various diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity remained unknown.

Double-stranded RNA (dsRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA induced silencing complex (RISC), which requires degradation of the sense strand of the dsRNA duplex. Sense strand was known to act as the first RISC substrate that is cleaved by Argonaute 2 in the middle of the duplex region. Immediately after the cleaved 5′-end and 3′-end fragments of the sense strand are removed from the endonuclease Ago2, the RISC becomes activated by the antisense strand (Rand et al. (2005) Cell 123, 621).

It was believed that when the cleavage of the sense strand is inhibited, the endonucleolytic cleavage of target mRNA is impaired (Leuschner et al. (2006) EMBO Rep., 7, 314; Rand et al. (2005) Cell 123, 621; Schwarz et al. (2004) Curr. Biol. 14, 787). Leuschner et al. showed that incorporation of a 2′-O-Me ribose to the Ago2 cleavage site in the sense strand inhibits RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep., 7, 314). A similar effect was observed with phosphorothioate modifications, showing that cleavage of the sense strand was required for efficient RNAi also in mammals.

Morrissey et al. used a siRNA duplex containing 2′-F modified residues, among other sites and modifications, also at the Ago2 cleavage site, and obtained compatible silencing compared to the unmodified siRNAs (Morrissey et al. (2005) Hepatology 41, 1349). However, Morrissey's modification is not motif specific, e.g., one modification includes 2′-F modifications on all pyrimidines on both sense and antisense strands as long as pyrimidine residue is present, without any selectivity; and hence it is uncertain, based on these teachings, if specific motif modification at the cleavage site of sense strand can have any actual effect on gene silencing activity.

Muhonen et al. used a siRNA duplex containing two 2′-F modified residues at the Ago2 cleavage site on the sense or antisense strand and found it was tolerated (Muhonen et al. (2007) Chemistry & Biodiversity 4, 858-873). However, Muhonen's modification is also sequence specific, e.g., for each particular strand, Muhonen only modifies either all pyrimidines or all purines, without any selectivity.

Choung et al. used a siRNA duplex containing alternative modifications by 2′-OMe or various combinations of 2′-F, 2′-OMe and phosphorothioate modifications to stabilize siRNA in serum to Sur10058 (Choung et al. (2006) Biochemical and Biophysical Research Communications 342, 919-927). Choung suggested that the residues at the cleavage site of the antisense strand should not be modified with 2′-OMe in order to increase the stability of the siRNA.

There is thus an ongoing need for iRNA duplex agents to improve the gene silencing efficacy of siRNA gene therapeutics. This invention is directed to that need.

SUMMARY

This invention provides effective nucleotide or chemical motifs for dsRNA agents optionally conjugated to at least one ligand, which are advantageous for inhibition of target gene expression, as well as RNAi compositions suitable for therapeutic use.

The inventors surprisingly discovered that introducing one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of a dsRNA agent that is comprised of modified sense and antisense strands enhances the gene silencing activity of the dsRNA agent.

In one aspect, the invention relates to a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The dsRNA duplex is represented by formula (III):

(III) sense: 5′ n_(p) -N_(a) -(X X X ) _(i)-N_(b)-Y Y Y -N_(b) -(Z Z Z)_(j) -N_(a)- n_(q) 3′, antisense: 3′ n_(p)'-N_(a)'-(X'X'X')_(k)-N_(b)'-Y'Y'Y'-N_(b)'-(Z'Z'Z')_(l)-N_(a)- n_(q)'5′ In formula (III), i, j, k, and 1 are each independently 0 or 1; p and q are each independently 0-6; n represents a nucleotide; each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each n_(p) and n_(q) independently represents an overhang nucleotide sequence comprising 0-6 nucleotides; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; wherein the modifications on Nb is different than the modification on Y and the modifications on Nb′ is different than the modification on Y′. At least one of the Y nucleotides forms a base pair with its complementary Y′ nucleotides, and wherein the modification on the Y nucleotide is different than the modification on the Y′ nucleotide.

Each n_(p) and n_(q) independently represents an overhang nucleotide sequence comprising 0-6 nucleotides; each n and n′ represents an overhang nucleotide; and p and q are each independently 0-6.

In another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at or near cleavage site by at least one nucleotide. The modification in the motif occurring at or near the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand.

In another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In another aspect, the invention further provides a method for delivering the dsRNA to a specific target in a subject by subcutaneous or intravenenuous administration.

DETAILED DESCRIPTION

A superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand and/or antisense strand of a dsRNA agent, particularly at or near the cleavage site. The sense strand and antisense strand of the dsRNA agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense and/or antisense strand. The dsRNA agent optionally conjugates with a GalNAc derivative ligand, for instance on the sense strand. The resulting dsRNA agents present superior gene silencing activity.

The inventors surprisingly discovered that having one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a dsRNA agent superiorly enhanced the gene silencing activity of the dsRNA agent.

Accordingly, the invention provides a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand. Each strand of the dsRNA agent can range from 12-30 nucleotides in length. For example, each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex dsRNA. The duplex region of a dsRNA agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-30 nucleotides in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

In one embodiment, the dsRNA agent of the invention comprises may contain one or more overhang regions and/or capping groups of dsRNA agent at the 3′-end, or 5′-end or both ends of a strand. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the dsRNA agent of the invention can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F 2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the dsRNA agent of the invention may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The dsRNA agent of the invention comprises only single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability. For example, the single-stranded overhang is located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The dsRNA may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the dsRNA agent of the invention may also have two blunt ends, at both ends of the dsRNA duplex.

In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 19 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 21 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the dsRNA agent of the invention comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end, wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3′-end of the antisense. Optionally, the dsRNA further comprises a ligand (preferably GalNAc3).

In one embodiment, the dsRNA agent of the invention comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the dsRNA agent of the invention comprising a sense and antisense strands, wherein said dsRNA agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein said 3′ end of said first strand and said 5′ end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and said second strand is sufficiently complementary to a target mRNA along at least 19 nt of said second strand length to reduce target gene expression when said dsRNA agent is introduced into a mammalian cell, and wherein dicer cleavage of said dsRNA preferentially results in an siRNA comprising said 3′ end of said second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNA agent further comprises a ligand.

In one embodiment, the sense strand of the dsRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the dsRNA agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For dsRNA agent having a duplex region of 17-23 nt in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1^(st) nucleotide from the 5′-end of the antisense strand, or, the count starting from the 1^(st) paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNA from the 5′-end.

The sense strand of the dsRNA agent comprises at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides of the motifs from both strands may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the dsRNA agent comprises more than one motif of three identical modifications on three consecutive nucleotides. The first motif should occur at or near the cleavage site of the strand and the other motifs may be a wing modifications. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide the chemistries of the motifs can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, the wing modifications may both occur at one end of the duplex region relative to the first motif which is at or near the cleavage site or each of the wing modifications may occur on either side of the first motif.

Like the sense strand, the antisense strand of the dsRNA agent comprises at least two motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that is present on the sense strand.

In one embodiment, the wing modification on the sense strand, antisense strand, or both strands of the dsRNA agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand, antisense strand, or both strands of the dsRNA agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the dsRNA agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the dsRNA agent each contain at least two wing modifications, the sense strand and the antisense strand can be aligned so that two wing modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides.

In one embodiment, every nucleotide in the sense strand and antisense strand of the dsRNA agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking 0 of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. E.g., a phosphorothioate modification at a non-linking 0 position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the sense strand and antisense strand each contains two differently modified nucleotides selected from 2′-O-methyl or 2′-fluoro.

In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxyfluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of an alternating pattern. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications of an alternating pattern. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In one embodiment, the dsRNA agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisenese strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In one embodiment, the dsRNA agent of the invention comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.

In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) . . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_(a)” and “N_(b)” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_(a) and N_(b) can be the same or different modifications. Alternatively, N_(a) and/or N_(b) may be present or absent when there is a wing modification present.

The dsRNA agent of the invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In one embodiment, the dsRNA comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In one embodiment the sense strand of the dsRNA comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5 or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment the antisense strand of the dsRNA comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3 or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.

In one embodiment, the dsRNA of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense and/or antisense strand.

In one embodiment, the dsRNA of the invention further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense and/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s).

In one embodiment, the dsRNA of the invention further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end), and one phosphorothioateinternucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA of the invention further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the dsRNA agent of the invention comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In one embodiment, the sense strand sequence may be represented by formula (I):

(I) 5′ n_(p)-N_(a)-(X X X )_(i)-N_(b)-Y Y Y -N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNA agent has a duplex region of 17-23 nucleotide pairs in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of − the sense strand, the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

(Ia) 5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ib) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′; or (Ic) 5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6 Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In one embodiment, the antisense strand sequence of the dsRNA may be represented by formula (II):

(II) 5′ n_(q)'-N_(a)'-(Z’Z'Z')_(k)-N_(b)'-Y'Y'Y'-N_(b)'-(X'X'X')₁-N'_(a)- n_(p)' 3′ wherein:

k and l are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide comprising 0-6 nucleotides;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNA agent has a duplex region of 17-23 nt in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1^(st) nucleotide, from the 5′-end; or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and 1 is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

(IIa) 5′ n_(q)'-N_(a)'-Z'Z'Z'-N_(b)'-Y'Y'Y'-N_(a)'-n_(p)'3′; (IIb) 5′ n_(q)'-N_(a)'-Y'Y'Y'-N_(b)'-X'X'X'-n_(p)'3′; or (IIc) 5′ n_(q)'-N_(a)'-Z'Z'Z'-N_(b)'-Y'Y'Y'-N_(b)'-X'X'X'-N_(a)'-n_(p)'3′.

When the antisense strand is represented by formula (IIa), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the dsRNA agent comprises YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib) and (Ic) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb) and (IIc), respectively.

Accordingly, the dsRNA agent may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (III):

(III) sense: 5′ n_(p) -N_(a)- (X X X)_(i) -N_(b)- Y Y Y -N_(b) -(Z Z Z)_(j)-N_(a)- n_(q) 3′ antisense: 3′ n_(p)'-N_(a)'-(X'X'X')_(k)-N_(b)'-Y'Y'Y'-N_(b)'-(Z'Z'Z')_(l)-N_(a)'- n_(q)' 5′

wherein:

j, k, and 1 are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q) independently represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is 1; or both k and l are 1.

In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (V):

(V) sense: 5′ N_(a)-(X X X)_(i)-N_(b)-Y Y Y -N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′ antisense: 3′ n_(p)'-N_(a)'-(X'X'X')_(k)-N_(b)'-Y'Y'Y'-N_(b)'-(Z'Z'Z')_(l)- N_(a)' 5′

wherein:

j, k, and l are each independently 0 or 1;

p and q are each independently 2;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, and n_(q) independently represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is 1; or both k and l are 1.

In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (Va):

(Va) sense: 5′ N_(a)-(X X X)_(i)-N_(b)-Y Y Y -N_(b)-(Z Z Z)_(j)-N_(a) 3′ antisense: 3′ n_(p)'-N_(a)'-(X'X'X')_(k)-N_(b)'-Y'Y'Y'-N_(b)'-(Z'Z'Z')_(l)- N_(a)' 5′

wherein:

j, k, and l are each independently 0 or 1;

p and q are each independently 2;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein

n_(p)′ represents an overhang nucleotide sequence; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

Exemplary combinations of the sense strand and antisense strand forming a dsRNA duplex include the formulas below:

(IIIa) 5′ n_(p) -N_(a) -Y Y Y -N_(b) -Z Z Z -N_(a) -n_(q) 3′ 3′ n_(p)'-N_(a)'-Y'Y'Y'-N_(b)'-Z'Z'Z'-N_(a)'n_(q)' 5′ (IIIb) 5′ n_(p)-N_(a)- X X X -N_(b) -Y Y Y - N_(a)-n_(q) 3′ 3′ n_(p)'-N_(a)'-X'X'X'-N_(b)'-Y'Y'Y'-N_(a)'-n_(q)' 5′ (IIIc) 5′ n_(p) -N_(a) -X X X -N_(b)-Y Y Y -N_(b)- Z Z Z -N_(a)-n_(q) 3′ 3′ n_(p)'-N_(a)'-X'X'X'-N_(b)'-Y'Y'Y'-N_(b)'-Z'Z'Z'-N_(a)-n_(q)' 5′

When the dsRNA agent is represented by formula (IIIa), each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIb), each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNA agent is represented as formula (IIIc), each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b), independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIIc) may be the same or different from each other.

When the dsRNA agent is represented by formula (III), (IIIa), (IIIb) or (IIIc), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

It is understood that N_(a) nucleotides from base pair with N_(a)′, N_(b) nucleotides from base pair with N_(b)′, X nucleotides from base pair with X′, Y nucleotides from base pair with Y′, and Z nucleotides from base pair with Z′.

When the dsRNA agent is represented by formula (IIIa) or (IIIc), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNA agent is represented as formula (IIIb) or (IIIc), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In one embodiment, the dsRNA agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), wherein said duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, said multimer further comprise a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.

In one embodiment, the dsRNA agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), wherein said duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, said multimer further comprises a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.

In one embodiment, two dsRNA agent represented by formula (III), (IIIa), (IIIb) or (IIIc) are linked to each other at the 5′ end, and one or both of the 3′ ends of the are optionally conjugated to a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.

Various publications described multimeric siRNA and can all be used with the dsRNA of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 which are hereby incorporated by their entirely.

The dsRNA agent that contains conjugations of one or more carbohydrate moieties to a dsRNA agent can optimize one or more properties of the dsRNA agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the dsRNA agent. E.g., the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

In embodiment the dsRNA of the invention is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the invention may optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end or both ends. For instance, the ligand may be conjugated to the sense strand, in particular, the 3′-end of the sense strand.

Ligands

A wide variety of entities can be coupled to the oligonucleotides of the present invention. Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether.

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of the molecule into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Ligands providing enhanced affinity for a selected target are also termed targeting ligands.

Some ligands can have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

Ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer. Table 2 shows some examples of targeting ligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelator (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake of the oligonucleotide into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL) and high-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 1). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 2)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO: 3) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO: 4) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing α_(V)B₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type lignads that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, or an apatamer. A cluster is a combination of two or more sugar units. The targeting ligands also include integrin receptor ligands, Chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligands can also be based on nucleic acid, e.g., an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Examplary PK modulator include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g. oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbaone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).

In addition, aptamers that bind serum components (e.g. serum proteins) are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S. patent application Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which are incorporated by reference in their entireties for all purposes.

When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In a preferred embodiment, all the ligands have different properties.

Ligands can be coupled to the oligonucleotides at various places, for example, 3′-end, 5′-end, and/or at an internal position. In preferred embodiments, the ligand is attached to the oligonucleotides via an intervening tether, e.g. a carrier described herein. The ligand or tethered ligand may be present on a monomer when said monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated via coupling to a “precursor” monomer after said “precursor” monomer has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporated into a growing oligonucleotide strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having complementary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.

For double-stranded oligonucleotides, ligands can be attached to one or both strands. In some embodiments, a double-stranded iRNA agent contains a ligand conjugated to the sense strand. In other embodiments, a double-stranded iRNA agent contains a ligand conjugated to the antisense strand.

In some embodiments, ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic linkages of nucleic acid molecules. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2′, 3′, and 5′ carbon atoms. The 1′ position can also be attached to a conjugate moiety, such as in an abasic residue. Internucleosidic linkages can also bear conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used, although the ligand is typically a carbohydrate e.g. monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to a bivalent and trivalent branched linkers include the structures shown in any of formula (IV)-(VII):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q^(4A), q^(4B), q^(5A), q^(5B) and q^(5C) represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (VII):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the following compounds:

Definitions

As used herein, the terms “dsRNA”, “siRNA”, and “iRNA agent” are used interchangeably to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an siRNA agent of 21 to 23 nucleotides.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, a dsRNA agent of the invention is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the dsRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the dsRNA agent of the invention is “exactly complementary” to a target RNA, e.g., the target RNA and the dsRNA duplex agent anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the dsRNA agent of the invention specifically discriminates a single-nucleotide difference. In this case, the dsRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) for example of length less than 100, 200, 300, or 400 nucleotides.

The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine. The term “alkyl” refers to saturated and unsaturated non-aromatic hydrocarbon chains that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation propyl, allyl, or propargyl), which may be optionally inserted with N, O, or S. For example, C₁-C₁₀ indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. The term “alkoxy” refers to an —O-alkyl radical. The term “alkylene” refers to a divalent alkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene. The term “aminoalkyl” refers to an alkyl substituted with an amino The term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

The term “substituted” refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkyl sulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, aryl aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C₅ and above (preferably C₅-C₈ sugars; di- and trisaccharides include sugars having two or three monosaccharide units (preferably C₅-C₈).

Alternative Embodiments

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the antisense strand. Every nucleotide in the sense strand and antisense strand has been modified. The modifications on sense strand and antisense strand each independently comprises at least two different modifications.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the antisense strand. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides. The modification pattern of the antisense strand is shifted by one or more nucleotides relative to the modification pattern of the sense strand.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, when at least one of the motifs occurs at the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at or near cleavage site by at least one nucleotide.

In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at or near cleavage site by at least one nucleotide. The modification in the motif occurring at the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand. In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 12 to 30 nucleotides. The sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at the cleavage site in the strand. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides.

The sense strand may further comprises one or more motifs of three identical modifications on three consecutive nucleotides, where the one or more additional motifs occur at another portion of the strand that is separated from the three 2′-F modifications at the cleavage site by at least one nucleotide. The antisense strand may further comprises one or more motifs of three identical modifications on three consecutive nucleotides, where the one or more additional motifs occur at another portion of the strand that is separated from the three 2′-O-methyl modifications by at least one nucleotide. At least one of the nucleotides having a 2′-F modification may form a base pair with one of the nucleotides having a 2′-O-methyl modification.

In one embodiment, the dsRNA of the invention is administered in buffer.

In one embodiment, siRNA compounds described herein can be formulated for administration to a subject. A formulated siRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein. For example, in particular embodiments the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

A siRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a siRNA, e.g., a protein that complexes with siRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the siRNA preparation includes another siNA compound, e.g., a second siRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species. Such siRNAs can mediate RNAi with respect to a similar number of different genes.

In one embodiment, the siRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA). For example, a siRNA composition for the treatment of a viral disease, e.g., HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a siRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

Exemplary formulations are discussed below.

Liposomes.

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNAs, and such practice is within the invention. An siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the siRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the siRNA are delivered into the cell where the siRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the siRNA to particular cell types.

A liposome containing a siRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The siRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the siRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of siRNA.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging siRNA preparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver siRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated siRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of siRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer siRNA, into the skin. In some implementations, liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with siRNA are useful for treating a dermatological disorder.

Liposomes that include siRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include siRNA can be delivered, for example, subcutaneously by infection in order to deliver siRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

Surfactants.

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNA compounds, and such practice is within the scope of the invention. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). siRNA (or a precursor, e.g., a larger dsiRNA which can be processed into a siRNA, or a DNA which encodes a siRNA or precursor) compositions can include a surfactant. In one embodiment, the siRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and other Membranous Formulations.

For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNA compounds, and such practice is within the invention. The siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

Particles.

For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these particles, formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the invention. In another embodiment, an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

Pharmaceutical Compositions

The iRNA agents of the invention may be formulated for pharmaceutical use. Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the dsRNA agents in any of the preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.

The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.

The phrase “therapeutically-effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present invention.

iRNA agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

Double-stranded RNAi agents are produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of a dsRNA agent and one that produces a transcript that includes the bottom strand of a dsRNA agent. When the templates are transcribed, the dsRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.

Routes of Delivery

A composition that includes an iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

Dosage

In one aspect, the invention features a method of administering a dsRNA agent, e.g., a siRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the dsRNA agent, e.g., a siRNA agent, e.g., double stranded siRNA agent that (a) the double-stranded part is 14-30 nucleotides (nt) long, for example, 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application. In some embodiments dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight.

In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than a dsRNA agent, e.g., other than a siRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of a dsRNA agent, e.g., a siRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a dsRNA agent, e.g., a siRNA agent, (e.g., a precursor, e.g., a larger dsRNA agent which can be processed into a siRNA agent, or a DNA which encodes a dsRNA agent, e.g., a siRNA agent, or precursor thereof). The maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 15 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are, for example, administered no more than once every 2, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In certain embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

In one embodiment, the composition includes a plurality of dsRNA agent species. In another embodiment, the dsRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of dsRNA agent species is specific for different naturally occurring target genes. In another embodiment, the dsRNA agent is allele specific.

The dsRNA agents of the invention described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the dsRNA agent, e.g., a siRNA agent, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of dsRNA agents described herein

Methods of Inhibiting Expression of the Target Gene

Embodiments of the invention also relate to methods for inhibiting the expression of a target gene. The method comprises the step of administering the dsRNA agents in any of the preceding embodiments, in an amount sufficient to inhibit expression of the target gene.

Another aspect the invention relates to a method of modulating the expression of a target gene in a cell, comprising providing to said cell a dsRNA agent of this invention. In one embodiment, the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene, mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES Example 1. In Vitro Screening of siRNA Duplexes Cell Culture and Transfections:

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done using 8, 4 fold serial dilutions with a maximum dose of 10 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂O per reaction were added into 5 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)), 0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914_m1 (human) cat #Rn00562124_m1 (rat)) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plate (Roche cat #04887301001). Real time PCR was done in a Roche LC 480 Real Time PCR machine (Roche). Each duplex was tested in at least two independent transfections and each transfection was assayed in duplicate, unless otherwise noted.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. IC₅₀s were calculated for each individual transfection as well as in combination, where a single IC₅₀ was fit to the data from both transfections.

The results of gene silencing of the exemplary siRNA duplex with various motif modifications of the invention are shown in the table below.

Example 2. RNA Synthesis and Duplex Annealing

1. Oligonucleotide Synthesis:

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer or an ABI 394 synthsizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis unless otherwise specified. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from (Promega). All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which was used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M, American International Chemicals), for the PO-oxidation Iodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using solid support containing the corresponding ligand. For example, the introduction of carbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of a sequence was achieved by starting the synthesis with the corresponding carbohydrate solid support. Similarly a cholesterol moiety at the 3′-end was introduced by starting the synthesis on the cholesterol support. In general, the ligand moiety was tethered to trans-4-hydroxyprolinol via a tether of choice as described in the previous examples to obtain a hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety was then coupled to a solid support via a succinate linker or was converted to phosphoramidite via standard phosphitylation conditions to obtain the desired carbohydrate conjugate building blocks. Fluorophore labeled siRNAs were synthesized from the corresponding phosphoramidite or solid support, purchased from Biosearch Technologies. The oleyl lithocholic (GalNAc)₃ polymer support made in house at a loading of 38.6 μmol/gram. The Mannose (Man)₃ polymer support was also made in house at a loading of 42.0 μmol/gram.

Conjugation of the ligand of choice at desired position, for example at the 5′-end of the sequence, was achieved by coupling of the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions unless otherwise specified. An extended 15 min coupling of 0.1M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate was carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate was introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent The cholesterol phosphoramidite was synthesized in house, and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite was 16 minutes.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 ml glass bottle (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C. The bottle was cooled briefly on ice and then the ethanolic ammonia mixture was filtered into a new 250 ml bottle. The CPG was washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture was then reduced to ˜30 ml by roto-vap. The mixture was then frozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction was then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to 6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified reverse phase preparative HPLC. The unconjugated oligonucleotides were purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers were 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotides were diluted in water to 150 μl and then pipetted in special vials for CGE and LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. siRNA Preparation

For the preparation of siRNA, equimolar amounts of sense and antisense strand were heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex was confirmed by HPLC analysis.

TABLE 2 ANGPTL3 modified duplex Sense strand (S) Antisense strand (AS) (SEQ ID NOS 5-424, (SEQ ID NOS 425-844, respectively, respectively, % of miRNA remained Duplex in order in order conc. of siRNA ID S ID of appearance) AS ID of appearance) 1 nM 0.1 nM 0.01 nM IC50 (nM) D1000 S1000 AfuGfuAfaCfcAfAfGfa AS1000 AfUfgGfaAfuAfcUfcuuGfg 0.03 0.1 0.47 0.006 GfuAfuUfcCfasu UfufuAfcAfusGfsa D1001 S1001 AfsuGfuAfaCfcAfAfGf AS1001 aUfsgGfAfAfuAfcUfcuuGf 0.03 0.10 0.49 0.0065 aGfuAfuucCfasUf gUfuAfcAfusGfsa D1002 S1002 AfuGfuAfaCfcAfAfGfa AS1002 aUfgGfAfAfuAfcUfcuuGfg 0.04 0.10 0.46 0.0068 GfuAfuucCfasUf sUfuAfcAfusGfsa D1003 S1003 AfuGfuAfaCfcAfAfGfa AS1003 aUfgGfAfAfuAfcUfcuuGfg 0.05 0.12 0.56 0.0073 GfuAfuucCfasUf UfsuAfcAfusGfsa D1004 S1004 aUGuaACccAGagUAuuCC AS1004 AUggAAuaCUcuUGguUAcaUs 0.07 0.13 0.44 0.008 asu Gsa D1005 S1005 AfuGfuAfaCfcAfAfGfa AS1005 aUfgGfAfAfuAfcUfcuuGfg 0.06 0.11 0.53 0.0093 GfuAfuucCfasUf sUfsuAfcAfusGfsa D1006 S1006 AfuGfuAfAfccAfAfGfa AS1006 aUfgGfaAfuAfcUfcuuGfGf 0.05 0.16 0.55 0.0095 GfuAfuUfcCfasUf uuAfcAfusGfsa D1007 S1007 AfuGfuAfAfCfcAfAfGf AS1007 aUfgGfaAfuAfcUfcuuGfgu 0.05 0.14 0.48 0.0098 aGfuAfuUfcCfasUf uAfcAfusGfsa D1008 S1008 auguaaccaadGadGudAu AS1008 aUfgGfaAfuAfcUfcUfuGfg 0.07 0.11 0.33 0.010 dAcdGasu UfuAfcAfusGfsa D1009 S1009 UfgGfGfAfuUfuCfAfUf AS1009 uCfuugGfuUfaCfaugAfaAf 0.03 0.14 0.56 0.0101 gUfaAfcCfAfAfgsAf uccCfasUfsc D1010 S1010 UfgGfgauUfuCfAfUfgU AS1010 uCfuUfgGfuUfaCfaugAfaA 0.03 0.14 0.65 0.0101 faAfcCfaAfgsAf fUfCfcCfasUfsc D1011 S1011 aUfGfuAfAfccAfAfGfa AS1011 aUfgGfaAfuAfcUfcuuGfGf 0.06 0.10 0.55 0.011 GfuAfuUfcCfasUf uuAfcaUfsgsa D1012 S1012 UfgGfgAfuUfuCfAfUfg AS1012 uCfuUfgGfUfUfaCfaugAfa 0.04 0.13 0.54 0.0114 UfaacCfaAfgsAf AfuCfcCfasUfsc D1013 S1013 auguaaccaadGadGudAu AS1013 aUfgGfaAfuAfcUfcUfugdG 0.11 0.19 0.49 0.011 dAcdGasu udTadCadTsgsa D1014 S1014 AfuGfuaaCfcAfAfGfaG AS1014 aUfgGfaAfuAfcUfcuuGfgU 0.04 0.16 0.59 0.013 fuAfuUfcCfasUf fUfAfcAfusGfsa D1015 S1015 AfuguAfaccAfaGfdAGf AS1015 dAUdGgdAadTAfdCUfcUfuG 0.07 0.15 0.51 0.013 dTAdTudCcdAsu fgUfuAfcAfusGfsa D1016 S1016 auGfuAfaCfcAfAfGfaG AS1016 aUfgGfaAfuAfcUfcuuGfgU 0.05 0.14 0.64 0.013 fuAfuUfcCfasUf fuAfcAfUfsGfsa D1017 S1017 UfGfggAfuUfuCfAfUfg AS1017 uCfuUfgGfuuaCfaugAfaAf 0.09 0.41 0.74 0.0133 UfAfAfcCfaAfgsAf uCfCfcasUfsc D1018 S1018 AfuguAfaCfcAfAfGfaG AS1018 aUfgGfaAfuAfcUfcuuGfgU 0.03 0.14 0.61 0.014 fuAfuUfcCfasUf fuAfCfAfusGfsa D1019 S1019 AfuGfuAfaccAfAfGfaG AS1019 aUfgGfaAfuAfcUfcuuGfGf 0.02 0.2 0.7 0.014 fuAfuUfcCfasUf UfuAfcAfusGfsa D1020 S1020 AfsuGfuAfaCfcAfAfGf AS1020 asUfsgGfAfAfuAfcUfcuuG 0.04 0.16 0.67 0.0156 aGfuAfuucCfasUf fgUfuAfcAfusGfsa D1021 S1021 aUfguAfAfccAfAfgagU AS1021 aUfGfgAfaUfaCfUfCfuuGf 0.11 0.24 0.64 0.016 faUfuCfcasUf GfuuAfCfaUfsgsa D1022 S1022 dTdGggdAdTuudCdAugd AS1022 udCdTugdGdTuadCdAugdAd 0.08 0.27 0.64 0.0161 TdAacdCdAagsdA AaudCdCcasdTsc D1023 S1023 AfsuGfuAfaCfcAfAfGf AS1023 aUfgsGfAfAfuAfcUfcuuGf 0.03 0.19 0.63 0.0163 aGfuAfuucCfasUf gUfuAfcAfusGfsa D1024 S1024 UfgGfgAfuUfuCfAfUfg AS1024 uCfuUfgGfuUfAfCfaugAfa 0.05 0.25 0.69 0.0164 uaAfcCfaAfgsAf AfuCfcCfasUfsc D1025 S1025 UfgGfgAfuUfuCfAfUfg AS1025 uCfuUfgGfuuaCfaugAfaAf 0.04 0.18 0.75 0.0166 UfAfAfcCfaAfgsAf uCfcCfasUfsc D1026 S1026 UfgGfgAfuUfuCfAfUfg AS1026 uCfuUfgGfuUfaCfaugAfaA 0.04 0.19 0.66 0.0178 UfaAfcCfaAfgsAf fuCfcCfasUfsc D1027 S1027 UfgGfgAfuUfuCfAfUfg AS1027 uCfuUfGfGfuUfaCfaugAfa 0.04 0.19 0.69 0.018 UfaAfccaAfgsAf AfuCfcCfasUfsc D1028 S1028 dAdTgudAdAccdAdAgad AS1028 adTdGgadAdTacdTdCuudGd 0.15 0.29 0.72 0.018 GdTaudTdCcasdT GuudAdCausdGsa D1029 S1029 AdTGdTAdACdCAdAGdAG AS1029 dAUdGGdAAdTAdCUdCUdTGd 0.1 0.27 0.61 0.018 dTAdTUdCCdAsU GUdTAdCAdTsGsdA D1030 S1030 UfgGfGfAfuuuCfAfUfg AS1030 uCfuUfgGfuUfaCfaugAfAf 0.04 0.21 0.64 0.0187 UfaAfcCfaAfgsAf AfuccCfasUfsc D1031 S1031 AfuGfuAfAfccAfAfGfA AS1031 AfUfGfGfAfAfuAfCfUfCfU 0.06 0.15 0.62 0.019 fGfuAfuuccAfsu fuGfGfuuAfcAfusGfsa D1032 S1032 AfsuGfuAfaCfcAfAfGf AS1032 asUfgGfAfAfuAfcUfcuuGf 0.09 0.34 0.78 0.021 aGfuAfuucCfasUf gUfsuAfcAfusGfsa D1033 S1033 UfgGfgAfuUfuCfaUfGf AS1033 uCfuUfgGfUfUfacaUfgAfa 0.06 0.26 0.57 0.0212 UfaacCfaAfgsAf AfuCfcCfasUfsc D1034 S1034 AfuGfuAfAfccAfaGfaG AS1034 aUfgGfaAfuAfcUfcUfuGfG 0.11 0.39 0.82 0.0216 fuAfuUfcCfasUf fuuAfcAfusGfsa D1035 S1035 UfgGfgAfuuuCfAfUfgU AS1035 uCfuUfgGfuUfaCfaugAfAf 0.04 0.16 0.56 0.0222 faAfcCfaAfgsAf AfuCfcCfasUfsc D1036 S1036 UfgGfGfAfuUfuCfaUfg AS1036 uCfuugGfuUfaCfaUfgAfaA 0.06 0.31 0.78 0.0234 UfaAfcCfAfAfgsAf fuccCfasUfsc D1037 S1037 UfgGfGfAfuUfuCfAfUf AS1037 uCfuUfgGfuUfaCfaugAfaA 0.03 0.14 0.62 0.0235 gUfaAfcCfaAfgsAf fuccCfasUfsc D1038 S1038 UfGfggAfUfuuCfAfugU AS1038 uCfUfugGfUfuaCfAfugAfA 0.09 0.39 0.78 0.0239 fAfacCfAfagsAf fauCfCfcasUfsc D1039 S1039 AfuGfuAfaCfcAfAfGfa AS1039 aUfgGfAfAfuAfcUfcuuGfg 0.03 0.14 0.59 0.025 GfuAfuucCfasUf UfuAfcAfusGfsa D1040 S1040 AfuGfuAfaCfcAfAfGfa AS1040 aUfGfGfaAfuAfcUfcuuGfg 0.03 0.13 0.56 0.025 GfuAfuUfccasUf UfuAfcAfusGfsa D1041 S1041 AfsuGfuAfaCfcAfAfGf AS1041 asUfgGfAfAfuAfcUfcuuGf 0.06 0.27 0.79 0.0252 aGfuAfuucCfasUf gUfuAfcAfusGfsa D1042 S1042 UfgGfgAfuuuCfAfUfgU AS1042 uCfuUfgGfuuaCfaugAfAfA 0.05 0.27 0.67 0.0259 fAfAfcCfaAfgsAf fuCfcCfasUfsc D1043 S1043 AfuGfuAfaCfcAfAfGfa AS1043 aUfgGfaAfUfAfcUfcuuGfg 0.02 0.16 0.63 0.027 GfuauUfcCfasUf UfuAfcAfusGfsa D1044 S1044 AfsuGfuAfaCfcAfAfGf AS1044 asUfgGfAfAfuAfcUfcuuGf 0.06 0.30 0.81 0.0271 aGfuAfuucCfasUf gsUfsuAfcAfusGfsa D1045 S1045 aUfguAfAfccAfAfgaGf AS1045 aUfGfgaAfUfacUfCfuuGfG 0.12 0.29 0.8 0.028 GfauUfCfcasUf fuuAfCfaUfsgsa D1046 S1046 AfuGfuAfaCfcAfAfGfa AS1046 aUfgGfaAfuAfCfUfcuuGfg 0.03 0.15 0.59 0.030 guAfuUfcCfasUf UfuAfcAfusGfsa D1047 S1047 UfgGfGfAfuUfuCfaUfg AS1047 uCfuUfgGfuuaCfaUfgAfaA 0.08 0.44 0.83 0.0324 UfAfAfcCfaAfgsAf fuccCfasUfsc D1048 S1048 AfuGfuAfaCfcAfAfGfa AS1048 aUfgGfaAfuAfcUfcuuGfgU 0.07 0.23 0.67 0.036 GfuAfuUfcCfasUf fuAfcAfusGfsa D1049 S1049 AfuGfuAfAfccAfAfGfA AS1049 AfUfGfGfAfAfuAfCfUfCfU 0.08 0.23 0.73 0.037 fGfuAfuuccAfsu fUfGfGfUfuAfCfAfusGfsa D1050 S1050 UfgGfgAfuuuCfaUfgUf AS1050 uCfuugGfuUfaCfaUfgAfAf 0.06 0.29 0.78 0.0372 aAfcCfAfAfgsAf AfuCfcCfasUfsc D1051 S1051 AfuGfuAfaccaagaguAf AS1051 aUfgGfaAfudAcdTcdTudGg 0.12 0.41 0.86 0.040 uUfcCfasUf dTuAfcAfusgsa D1052 S1052 AfuguAfaccAfaGfdAGf AS1052 aUfgGfaAfuAfcUfcUfuGfg 0.1 0.22 0.72 0.042 dTAdTUdCcdAsu UfuAfcAfusGfsa D1053 S1053 AfuguAfaccAfaGfdAGf AS1053 dAUdGGdAAfuAfcUfcUfuGf 0.09 0.31 0.69 0.044 dTAdTUdCcdAsu GfUfuAfCfAfusGfsa D1054 S1054 AfuGfuAfaCfcAfaGfad AS1054 adTdGGfaAfudAdCUfcUfuG 0.1 0.45 0.75 0.047 GdTAfuUfcdCdAsUf fgUfuAfcAfusGfsa D1055 S1055 AfuguAfaccAfaGfaGfd AS1055 dAUdGGdAadTAfcUfcUfuGf 0.12 0.26 0.7 0.049 TAdTUdCcdAsu gUfuAfcAfusGfsa D1056 S1056 AuGuAaCcAaGaGuAuUcC AS1056 aUgGaAuAcUcUuGgUuAcAus 0.08 0.24 0.65 0.050 asU Gsa D1057 S1057 AfuguAfaccAfagaGfua AS1057 aUfGfGfaAfUfAfcUfCfUfu 0.14 0.42 0.62 0.051 uUfccasUf GfGfUfuAfCfAfusGfsa D1058 S1058 AfuGfuAfaccaagaguAf AS1058 aUfgGfaAfudAcdTcdTudGg 0.12 0.36 0.86 0.053 uUfcCfasUf dTuAfcAfusGfsa D1059 S1059 AfuguAfaccAfaGfdAGf AS1059 dAUdGGdAadTAfdCUfcUfuG 0.09 0.27 0.7 0.054 dTAdTUdCcdAsu fgUfuAfcAfusGfsa D1060 S1060 adTgudAdAccdAdAgagd AS1060 adTdGgdAadTadCdTdCuudG 0.11 0.37 0.66 0.056 TadTudCcasdT dGuudAdCadTsgsa D1061 S1061 AfuGfuAfaCfcAfaGfdA AS1061 adTdGGfaAfuAfdCdTcUfuG 0.1 0.31 0.77 0.059 dGuAfuUfcdCdAsUf fgUfuAfcAfusGfsa D1062 S1062 AfuguAfaccAfaGfdAGf AS1062 aUfgGfaAfuAfcUfcUfuGfg 0.1 0.27 0.65 0.059 dTAdTudCcdAsu UfuAfcAfusGfsa D1063 S1063 adTdGuadAdCccdAdGag AS1063 dAdTggdAdAuadCdTcudTdG 0.12 0.44 0.82 0.064 dTdAuudCdCasu gudTdAcadTsdGsa D1064 S1064 AfuGfuAfaCfcAfaGfaG AS1064 adTdGGfaAfdTdAcUfcUfuG 0.12 0.32 0.83 0.064 fdTdAuUfcdCdAsUf fgUfuAfcAfusGfsa D1065 S1065 AfuguAfaccAfaGfaGfd AS1065 dAUdGgdAadTAfcUfcUfuGf 0.13 0.34 0.72 0.066 TAdTudCcdAsu gUfuAfcAfusGfsa D1066 S1066 AfuGfuAfaCfcAfaGfaG AS1066 adTdGGfadAdTAfcUfcUfuG 0.11 0.33 0.72 0.067 fudAdTUfcdCdAsUf fgUfuAfcAfusGfsa D1067 S1067 AfuguAfaccAfaGfaGfd AS1067 aUfgGfaAfuAfcUfcUfuGfg 0.11 0.37 0.62 0.070 TAdTUdCcdAsu UfuAfcAfusGfsa D1068 S1068 AfuguAfaccAfaGfaGfd AS1068 dAUdGGdAAuAfcUfcUfuGfG 0.16 0.33 0.64 0.072 TAdTUdCcdAsu fUfuAfCfAfusGfsa D1069 S1069 aUfGfuaAfCfccAfGfag AS1069 AfUfggAfAfuaCfUfcuUfGf 0.14 0.43 0.73 0.074 UfAfuuCfCfasu guUfAfcaUfsGfsa D1070 S1070 AfuGfuAfaCfCfAfaGfa AS1070 aUfgGfaAfuAfCfUfcUfugg 0.08 0.42 0.94 0.075 guAfuUfcCfasUf UfuAfcAfusGfsa D1071 S1071 UfgGfgAfuuuCfaUfgUf AS1071 uCfuUfgGfuUfaCfaUfgAfA 0.14 0.28 0.83 0.0797 aAfcCfaAfgsAf fAfuCfcCfasUfsc D1072 S1072 AfuGfuAfaCfcAfaGfAf AS1072 aUfgGfaAfUfAfcucUfuGfg 0.05 0.26 0.8 0.082 GfuauUfcCfasUf UfuAfcAfusGfsa D1073 S1073 AfuGfuAfaCfcAfaGfad AS1073 aUfgGfadAdTdAdCUfcUfuG 0.12 0.41 0.73 0.083 GdTdAdTUfcCfasUf fgUfuAfcAfusGfsa D1074 S1074 AfUfguAfAfccAfAfgaG AS1074 aUfGfgaAfUfacUfCfuuGfG 0.14 0.44 0.75 0.086 fUfauUfCfcasUf fuuAfCfausGfsa D1075 S1075 AfuGfuAfaCfcAfaGfaG AS1075 aUfgGfdAdAdTdAcUfcUfuG 0.1 0.41 0.72 0.088 fuAfuUfcCfasUf fgUfuAfcAfusGfsa D1076 S1076 AfuGfuAfaCfcAfaGfaG AS1076 aUfgdGdAdAdTAfcUfcUfuG 0.15 0.45 0.86 0.088 fudAdTdTdCCfasUf fgUfuAfcAfusGfsa D1077 S1077 AfuGfuAfaCfcAfaGfaG AS1077 AfUfgGfaAfuAfcUfcUfuGf 0.08 0.46 0.95 0.092 fuAfuUfcCfasu gUfuAfcAfusGfsa D1078 S1078 AfuGfuAfaCfcAfaGfaG AS1078 dAUdGGdAadTAfcUfcUfuGf 0.09 0.32 0.76 0.093 fuAfuUfcCfasUf gUfuAfcAfusGfsa D1079 S1079 AfuguAfaccAfaGfaGfd AS1079 dAudGgdAadTAfcUfcUfuGf 0.14 0.38 0.76 0.095 TadTudCcdAsu gUfuAfcAfusGfsa D1080 S1080 AfuGfuAfaCfcAfaGfAf AS1080 aUfgGfAfAfuAfcucUfuGfg 0.05 0.42 0.86 0.099 GfuAfuucCfasUf UfuAfcAfusGfsa D1081 S1081 AfuGfuAfaCfcAfaGfaG AS1081 dAdTdGdGaAfuAfcUfcUfuG 0.17 0.47 0.9 0.105 fuAfuUfdCdCdAsdT fgUfuAfcAfusGfsa D1082 S1082 AfuGfuAfaccaagaguAf AS1082 aUfgGfaAfudACfudCUfudG 0.12 0.44 0.83 0.106 uUfcCfasUf GfudTAfcAfusgsa D1083 S1083 AfuGfuAfaCfcAfaGfaG AS1083 adTdGGfaAfdTdAcUfcUfuG 0.11 0.34 0.74 0.109 fuAfuUfcCfasUf fgUfuAfcAfusGfsa D1084 S1084 AfuGfuAfAfCfcAfaGfa AS1084 aUfgGfaAfUfAfcUfcUfuGf 0.1 0.45 0.93 0.117 GfuauUfcCfasUf guuAfcAfusGfsa D1085 S1085 AfuGfUfAfaCfcAfaGfa AS1085 aUfgGfaAfUfAfcUfcUfuGf 0.07 0.42 0.78 0.120 GfuauUfcCfasUf gUfuacAfusGfsa D1086 S1086 aUfguAfAfccAfAfgaGf AS1086 aUfgGfaAfuAfcUfCfuuGfG 0.17 0.45 0.83 0.1197 uAfuUfcCfasUf fuuAfCfaUfsgsa D1087 S1087 AfuGfuAfaCfcAfaGfaG AS1087 AfUfgGfaAfuacUfcUfuGfg 0.05 0.3 0.7 0.120 fUfAfuUfcCfasu UfuAfcAfusGfsa D1088 S1088 AfuGfuAfaCfcAfaGfaG AS1088 aUfgGfaAfuAfcUfcUfuGfg 0.11 0.46 0.8 0.120 fuAfuUfcCfasUf UfuAfcAfusgsa D1089 S1089 AfuGfuAfaCfcAfaGfaG AS1089 aUfgGfaAfuacUfcUfuGfgU 0.14 0.49 0.85 0.122 fUfAfuUfcCfasUf fuAfcAfusGfsa D1090 S1090 AfuGfuAfaCfcAfaGfaG AS1090 aUfgGfaAfUfAfcUfcUfuGf 0.1 0.41 0.85 0.125 fuauUfcCfasUf gUfuAfcAfusGfsa D1091 S1091 AfuguAfaccAfaGfaGfd AS1091 aUfgGfaAfuAfcUfcUfuGfg 0.16 0.38 0.77 0.125 TAdTudCcdAsu UfuAfcAfusGfsa D1092 S1092 AfuGfuAfaCfcAfaGfAf AS1092 AfUfgGfaAfuAfcucUfuGfg 0.05 0.31 0.93 0.126 GfuAfuUfcCfasu UfuAfcAfusGfsa D1093 S1093 auGfuAfaCfcAfaGfAfG AS1093 aUfgGfaAfuAfcucUfuGfgU 0.06 0.33 0.9 0.135 fuAfuUfcCfasUf fuAfcAfUfsGfsa D1094 S1094 AfuGfuAfaCfcAfaGfaG AS1094 aUfGfGfaAfuacUfcUfuGfg 0.07 0.39 0.85 0.142 fUfAfuUfccasUf UfuAfcAfusGfsa D1095 S1095 AfuGfuAfaCfcAfaGfAf AS1095 aUfgGfaAfuAfcucUfuGfgU 0.09 0.39 0.76 0.146 GfuAfuUfcCfasUf fuAfcAfusGfsa D1096 S1096 AfuGfuAfaCfcAfaGfaG AS1096 aUfgGfAfAfuacUfcUfuGfg 0.06 0.38 0.85 0.147 fUfAfuucCfasUf UfuAfcAfusGfsa D1097 S1097 AfuGfUfAfaCfcAfaGfa AS1097 aUfgGfAfAfuAfcUfcUfuGf 0.12 0.47 0.87 0.147 GfuAfuucCfasUf gUfuacAfusGfsa D1098 S1098 AfuGfuAfaCfcAfaGfaG AS1098 aUfGfGfaauAfcUfcUfuGfg 0.06 0.42 0.85 0.151 fuAfUfUfccasUf UfuAfcAfusGfsa D1099 S1099 AfuGfuAfaCfcAfaGfaG AS1099 dAUdGGdAadTAfdCUfcUfuG 0.16 0.41 0.85 0.152 fuAfuUfcCfasUf fgUfuAfcAfusGfsa D1100 S1100 AfuguAfaccAfaGfaGfu AS1100 aUfgGfaAfuAfcUfcUfuGfg 0.15 0.48 0.72 0.152 AfuUfcCfasUf UfuAfcAfusGfsa D1101 S1101 AfuGfuAfaCfcAfaGfAf AS1101 aUfGfGfaAfuAfcucUfuGfg 0.06 0.38 0.94 0.158 GfuAfuUfccasUf UfuAfcAfusGfsa D1102 S1102 AfuGfuAfaccaagaguAf AS1102 aUfgGfaAfuAfdCuCfdTuGf 0.21 0.45 0.89 0.162 uUfcCfasUf 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AfcUfgUfusUfsu D1316 S1316 AfaCfaGfuGfuUfcUfuG AS1316 uUfAfUfaGfaGfcAfaGfaAf 0.07 0.16 0.53 fcUfcUfauasAf cAfcUfgUfususu D1317 S1317 AfacaGfuGfuUfcUfuGf AS1317 uUfaUfaGfaGfcAfaGfaAfc 0.07 0.16 0.55 cUfcUfaUfasAf AfcUfGfUfususu D1318 S1318 AfAfCfaGfuguUfcUfuG AS1318 uUfaUfaGfaGfcAfaGfaAfC 0.10 0.32 0.61 fcUfcUfaUfasAf fAfcUfguusUfsu D1319 S1319 AfaCfaGfuGfuUfcUfuG AS1319 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.16 0.53 fcUfcUfaUfasAf AfcUfgUfususu D1320 S1320 AfaCfaGfuGfuUfcUfuG AS1320 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.16 0.61 fcUfcUfaUfasAf AfcUfgUfususu D1321 S1321 AfaCfaGfuGfuUfcUfuG AS1321 uUfaUfagaGfcAfaGfaAfcA 0.06 0.14 0.58 fcUfCfUfaUfasAf fcUfgUfusUfsu D1322 S1322 AfaCfaGfuGfuUfcuuGf AS1322 uUfaUfaGfaGfcAfAfGfaAf 0.15 0.49 0.84 cUfcUfaUfasAf cAfcUfgUfusUfsu D1323 S1323 AfaCfaGfuGfuUfcUfuG AS1323 uUfaUfAfGfaGfcAfaGfaAf 0.07 0.20 0.62 fcUfcuaUfasAf cAfcUfgUfususu D1324 S1324 AfAfCfaGfuGfuUfcUfu AS1324 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.25 0.78 GfcUfcUfaUfasAf AfcUfguusUfsu D1325 S1325 AfAfCfaGfuGfuUfcUfu AS1325 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.18 0.80 GfcUfcUfaUfasAf AfcUfguusUfsu D1326 S1326 AfaCfaGfuGfuUfcUfuG AS1326 uUfauaGfaGfcAfaGfaAfcA 0.07 0.21 0.66 fcUfcUfAfUfasAf fcUfgUfusUfsu D1327 S1327 AfaCfaGfuGfuucUfuGf AS1327 uUfaUfaGfaGfcAfaGfAfAf 0.10 0.31 0.70 cUfcUfaUfasAf cAfcUfgUfusUfsu D1328 S1328 AfAfCfaGfuGfuUfcUfu AS1328 uUfAfUfaGfaGfcAfaGfaAf 0.07 0.15 0.55 GfcUfcUfauasAf cAfcUfguusUfsu D1329 S1329 AfaCfAfGfuGfuUfcUfu AS1329 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.19 0.71 GfcUfcUfaUfasAf AfcugUfusUfsu D1330 S1330 AfaCfaGfuGfuUfcUfuG AS1330 uuaUfaGfaGfcAfaGfaAfcA 0.09 0.27 0.76 fcUfcUfaUfAfsAf fcUfgUfusUfsu D1331 S1331 AfaCfaGfuguUfcUfuGf AS1331 uUfaUfaGfaGfcAfaGfaAfC 0.07 0.21 0.65 cUfcUfaUfasAf fAfcUfgUfusUfsu D1332 S1332 AfAfCfaGfuGfuUfcUfu AS1332 uUfaUfAfGfaGfcAfaGfaAf 0.07 0.17 0.53 GfcUfcuaUfasAf cAfcUfguusUfsu D1333 S1333 AfaCfaGfUfGfuUfcUfu AS1333 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.25 0.73 GfcUfcUfaUfasAf acUfgUfusUfsu D1334 S1334 AfaCfaguGfuUfcUfuGf AS1334 uUfaUfaGfaGfcAfaGfaAfc 0.07 0.18 0.54 cUfcUfaUfasAf AfCfUfgUfusUfsu D1335 S1335 AfaCfaGfuGfuUfcuuGf AS1335 uUfaUfaGfaGfcAfAfGfaAf 0.14 0.38 0.57 cUfcUfaUfasAf cAfcUfgUfususu D1336 S1336 AfaCfaGfuGfUfUfcUfu AS1336 uUfaUfaGfaGfcAfaGfaacA 0.16 0.50 0.96 GfcUfcUfaUfasAf fcUfgUfusUfsu D1337 S1337 AfaCfaGfuGfuUfcUfuG AS1337 uUfAfUfaGfaGfcAfaGfaAf 0.08 0.19 0.54 fcUfcUfauasAf cAfcUfgUfusUfsu D1338 S1338 AfAfCfaGfuGfuUfcUfu AS1338 uUfaUfaGfaGfCfAfaGfaAf 0.08 0.20 0.69 gcUfcUfaUfasAf cAfcUfguusUfsu D1339 S1339 AfaCfaGfuGfuUfCfUfu AS1339 uUfaUfaGfaGfcAfagaAfcA 0.07 0.16 0.55 GfcUfcUfaUfasAf fcUfgUfusUfsu D1340 S1340 AfaCfaGfuGfuUfcUfuG AS1340 uUfaUfAfGfaGfcAfaGfaAf 0.08 0.17 0.57 fcUfcuaUfasAf cAfcUfgUfusUfsu D1341 S1341 AfaCfaGfuguUfcUfuGf AS1341 uUfaUfaGfaGfcAfaGfaAfC 0.08 0.22 0.63 cUfcUfaUfasAf fAfcUfgUfususu D1342 S1342 AfAfCfaGfuGfuUfcuuG AS1342 uUfaUfaGfaGfcAfAfGfaAf 0.21 0.56 0.86 fcUfcUfaUfasAf cAfcUfguusUfsu D1343 S1343 AfacaGfuGfUfUfcUfuG AS1343 uUfaUfaGfaGfcAfaGfaacA 0.14 0.37 0.73 fcUfcUfaUfasAf fcUfGfUfusUfsu D1344 S1344 AfaCfaGfuGfuucUfUfG AS1344 uUfaUfaGfaGfcaaGfAfAfc 0.08 0.20 0.66 fcUfcUfaUfasAf AfcUfgUfusUfsu D1345 S1345 AfaCfAfGfuGfuUfcuuG AS1345 uUfaUfaGfaGfcAfAfGfaAf 0.12 0.34 0.73 fcUfcUfaUfasAf cAfcugUfusUfsu D1346 S1346 AfaCfaGfuGfUfUfcUfu AS1346 uUfAfUfaGfaGfcAfaGfaac 0.16 0.42 0.90 GfcUfcUfauasAf AfcUfgUfusUfsu D1347 S1347 AfaCfaGfuGfUfUfcUfu AS1347 uUfaUfaGfaGfcAfaGfaacA 0.17 0.43 0.85 GfcUfcUfaUfasAf fcUfgUfusUfsUf D1348 S1348 AfaCfAfGfuGfuucUfuG AS1348 uUfaUfaGfaGfcAfaGfAfAf 0.08 0.21 0.58 fcUfcUfaUfasAf cAfcugUfusUfsu D1349 S1349 AfaCfaGfuGfUfUfcUfu AS1349 uUfaUfAfGfaGfcAfaGfaac 0.21 0.39 0.88 GfcUfcuaUfasAf AfcUfgUfusUfsu D1350 S1350 AfaCfaguGfuUfcUfUfG AS1350 uUfaUfaGfaGfcaaGfaAfcA 0.06 0.13 0.52 fcUfcUfaUfasAf fCfUfgUfusUfsu D1351 S1351 AfaCfAfGfuguUfcUfuG AS1351 uUfaUfaGfaGfcAfaGfaAfC 0.08 0.21 0.58 fcUfcUfaUfasAf fAfcugUfusUfsu D1352 S1352 AfaCfaGfUfGfuUfcuuG AS1352 uUfaUfaGfaGfcAfAfGfaAf 0.18 0.49 0.84 fcUfcUfaUfasAf cacUfgUfusUfsu D1353 S1353 AfaCfaGfuGfUfUfcUfu AS1353 uUfaUfaGfAfGfcAfaGfaac 0.11 0.25 0.68 GfcucUfaUfasAf AfcUfgUfusUfsu D1354 S1354 AfacaGfuGfuUfcUfUfG AS1354 uUfaUfaGfaGfcaaGfaAfcA 0.07 0.15 0.52 fcUfcUfaUfasAf fcUfGfUfusUfsu D1355 S1355 AfaCfaGfUfGfuucUfuG AS1355 uUfaUfaGfaGfcAfaGfAfAf 0.10 0.26 0.63 fcUfcUfaUfasAf cacUfgUfusUfsu D1356 S1356 AfaCfaGfuGfUfUfcUfu AS1356 uUfaUfaGfaGfCfAfaGfaac 0.16 0.33 0.79 gcUfcUfaUfasAf AfcUfgUfusUfsu D1357 S1357 AfaCfAfGfuGfuUfcUfu AS1357 uUfaUfaGfaGfcAfaGfaAfc 0.09 0.19 0.51 GfcUfcUfaUfasAf AfcugUfusUfsUf D1358 S1358 AfaCfaGfuGfUfUfcuuG AS1358 uUfaUfaGfaGfcAfAfGfaac 0.22 0.48 0.71 fcUfcUfaUfasAf AfcUfgUfusUfsu D1359 S1359 AfaCfaGfuGfuUfcUfUf AS1359 uUfaUfaGfaGfcaaGfaAfcA 0.10 0.17 0.61 GfcUfcUfaUfasAf fcUfgUfusUfsUf D1360 S1360 AfaCfaguGfUfUfcUfuG AS1360 uUfaUfaGfaGfcAfaGfaacA 0.14 0.40 0.87 fcUfcUfaUfasAf fCfUfgUfusUfsu D1361 S1361 AfaCfaGfuGfuUfcUfUf AS1361 uUfaUfAfGfaGfcaaGfaAfc 0.07 0.14 0.52 GfcUfcuaUfasAf AfcUfgUfusUfsu D1362 S1362 aaCfaGfuGfuUfcUfuGf AS1362 uUfaUfaGfagcAfaGfaAfcA 0.10 0.28 0.81 CfUfcUfaUfasAf fcUfgUfUfsUfsu D1363 S1363 AfaCfaGfuGfuucUfuGf AS1363 uUfauaGfaGfcAfaGfAfAfc 0.06 0.16 0.68 cUfcUfAfUfasAf AfcUfgUfusUfsu D1364 S1364 AfaCfaGfuGfuUfcUfug AS1364 uuaUfaGfaGfCfAfaGfaAfc 0.09 0.26 0.67 cUfcUfaUfAfsAf AfcUfgUfusUfsu D1365 S1365 aacaguguucuugcucuau AS1365 uUfaUfaGfaGfcAfaGfaAfc 0.20 0.59 0.95 asa AfcUfgUfusUfsu D1366 S1366 AfaCfaGfuGfuUfcUfuG AS1366 uUfAfUfaGfagcAfaGfaAfc 0.06 0.13 0.53 fCfUfcUfauasAf AfcUfgUfusUfsu D1367 S1367 AfaCfaGfuGfuUfcUfuG AS1367 uUfaUfaGfagcAfaGfaAfcA 0.08 0.16 0.53 fCfUfcUfaUfasAf fcUfgUfusUfsUf D1368 S1368 AfaCfaGfuguUfcUfuGf AS1368 uUfauaGfaGfcAfaGfaAfCf 0.07 0.15 0.54 cUfcUfAfUfasAf AfcUfgUfusUfsu D1369 S1369 AfaCfaGfuGfuUfcuuGf AS1369 uuaUfaGfaGfcAfAfGfaAfc 0.23 0.56 0.89 cUfcUfaUfAfsAf AfcUfgUfusUfsu D1370 S1370 AfaCfaGfuGfuUfcUfuG AS1370 uUfaUfAfGfagcAfaGfaAfc 0.06 0.12 0.55 fCfUfcuaUfasAf AfcUfgUfusUfsu D1371 S1371 AfaCfaGfuGfuUfcUfuG AS1371 uUfaUfAfGfagcAfaGfaAfc 0.07 0.18 0.58 fCfUfcuaUfasAf AfcUfgUfusUfsu D1372 S1372 AfaCfaguGfuUfcUfuGf AS1372 uUfauaGfaGfcAfaGfaAfcA 0.06 0.15 0.56 cUfcUfAfUfasAf fCfUfgUfusUfsu D1373 S1373 AfaCfaGfuGfuucUfuGf AS1373 uuaUfaGfaGfcAfaGfAfAfc 0.21 0.51 0.89 cUfcUfaUfAfsAf AfcUfgUfusUfsu D1374 S1374 AfacaGfuguUfcUfuGfc AS1374 uUfaUfaGfaGfcAfaGfaAfC 0.08 0.21 0.64 UfcUfaUfasAf fAfcUfGfUfusUfsu D1375 S1375 AfaCfaGfuGfuUfcuuGf AS1375 uUfaUfaGfagcAfAfGfaAfc 0.15 0.40 0.94 CfUfcUfaUfasAf AfcUfgUfusUfsu D1376 S1376 AfaCfaGfuGfuUfcuuGf AS1376 uUfaUfaGfagcAfAfGfaAfc 0.13 0.40 0.96 CfUfcUfaUfasAf AfcUfgUfusUfsu D1377 S1377 AfaCfaGfuGfuUfcUfuG AS1377 uUfAfUfagaGfcAfaGfaAfc 0.08 0.17 0.64 fcUfCfUfauasAf AfcUfgUfusUfsu D1378 S1378 AfaCfaGfuguUfcUfuGf AS1378 uuaUfaGfaGfcAfaGfaAfCf 0.18 0.50 0.97 cUfcUfaUfAfsAf AfcUfgUfusUfsu D1379 S1379 AfaCfaGfuGfuucUfuGf AS1379 uUfaUfaGfagcAfaGfAfAfc 0.08 0.24 0.79 CfUfcUfaUfasAf AfcUfgUfusUfsu D1380 S1380 aaCfaGfuGfuUfcUfuGf AS1380 uUfauaGfaGfcAfaGfaAfcA 0.07 0.14 0.58 cUfcUfAfUfasAf fcUfgUfUfsUfsu D1381 S1381 AfaCfaguGfuUfcUfuGf AS1381 uuaUfaGfaGfcAfaGfaAfcA 0.11 0.34 0.96 cUfcUfaUfAfsAf fCfUfgUfusUfsu D1382 S1382 AfaCfaGfuguUfcUfuGf AS1382 uUfaUfaGfagcAfaGfaAfCf 0.08 0.18 0.69 CfUfcUfaUfasAf AfcUfgUfusUfsu D1383 S1383 AfaCfaGfuGfuUfcuuGf AS1383 uUfaUfagaGfcAfAfGfaAfc 0.14 0.38 0.85 cUfCfUfaUfasAf AfcUfgUfusUfsu D1384 S1384 AfaCfaGfuGfuUfcUfuG AS1384 uUfauaGfaGfcAfaGfaAfcA 0.07 0.16 0.54 fcUfcUfAfUfasAf fcUfgUfusUfsUf D1385 S1385 AfacaGfuGfuUfcUfuGf AS1385 uuaUfaGfaGfcAfaGfaAfcA 0.08 0.20 0.75 cUfcUfaUfAfsAf fcUfGfUfusUfsu D1386 S1386 aacaguguucUfuGfcUcU AS1386 uUfdAUdAGfaGfcAfaGfaad 0.25 0.56 0.90 audAsa CadCudGdTdTsusu D1387 S1387 AfaCfaguGfuUfcUfuGf AS1387 uUfaUfaGfagcAfaGfaAfcA 0.08 0.19 0.70 CfUfcUfaUfasAf fCfUfgUfusUfsu D1388 S1388 AfaCfaGfuGfuucUfuGf AS1388 uUfaUfagaGfcAfaGfAfAfc 0.08 0.14 0.60 cUfCfUfaUfasAf AfcUfgUfusUfsu D1389 S1389 AfaCfaGfuGfuUfcUfuG AS1389 uuaUfAfGfaGfcAfaGfaAfc 0.08 0.19 0.62 fcUfcuaUfAfsAf AfcUfgUfusUfsu D1390 S1390 aaCfaGfuGfuUfcUfuGf AS1390 uuaUfaGfaGfcAfaGfaAfcA 0.08 0.27 0.76 cUfcUfaUfAfsAf fcUfgUfUfsUfsu D1391 S1391 aacaguguucdTudGcdTc AS1391 uUfdAUdAGfaGfcAfaGfaad 0.18 0.36 0.81 dTadTasa CadCudGudTsusu D1392 S1392 AfacaGfuGfuUfcUfuGf AS1392 uUfaUfaGfagcAfaGfaAfcA 0.07 0.17 0.55 CfUfcUfaUfasAf fcUfGfUfusUfsu D1393 S1393 AfaCfaGfuguUfcUfuGf AS1393 uUfaUfagaGfcAfaGfaAfCf 0.07 0.15 0.57 cUfCfUfaUfasAf AfcUfgUfusUfsu D1394 S1394 AfaCfaGfuGfuUfcuuGf AS1394 uUfauaGfaGfcAfAfGfaAfc 0.26 0.68 1.06 cUfcUfAfUfasAf AfcUfgUfusUfsu D1395 S1395 AfaCfaGfuGfuUfcUfuG AS1395 uuaUfaGfAfGfcAfaGfaAfc 0.06 0.18 0.58 fcucUfaUfAfsAf AfcUfgUfusUfsu D1396 S1396 AfaCfaGfuGfuUfcUfuG AS1396 uuaUfaGfaGfcAfaGfaAfcA 0.09 0.27 0.73 fcUfcUfaUfAfsAf fcUfgUfusUfsUf D1397 S1397 AfaCfaAfuGfuUfcUfuG AS1397 uUfadTdAdGdAGfcAfaGfaG 0.20 0.51 0.73 fcdAdCdTdAUfasAf fcAfcAfgUfusUfsu D1398 S1398 AfacaGfuguUfcuuGfcu AS1398 uUfAfUfaGfAfGfcAfAfGfa 0.13 0.34 0.86 cUfauasAf AfCfAfcUfGfUfusUfsu D1399 S1399 dAacadGugudTcuudGcu AS1399 udTdAdTadGdAdGcdAdAdGa 0.24 0.42 0.82 cdTauasdA dAdCdAcdTdGdTusdTsu D1400 S1400 AfaCfaAfuGfuUfcUfuG AS1400 uUfaUfdAdGdAdGcAfaGfaG 0.49 0.85 0.78 fdCdAdCdTaUfasAf fcAfcAfgUfusUfsu D1401 S1401 AfaCfaAfuGfuUfcUfud AS1401 uUfaUfadGdAdGdCAfaGfaG 0.67 0.83 0.85 GdCdAdCUfaUfasAf fcAfcAfgUfusUfsu D1402 S1402 aaCfAfguGfUfucUfUfg AS1402 uUfaUfAfgaGfCfaaGfAfac 0.18 0.47 0.80 cUfCfuaUfAfsa AfCfugUfUfsusu D1403 S1403 AfaCfaAfuGfuUfcUfuG AS1403 udTdAUfadGdAGfcAfaGfaG 0.73 0.89 0.77 fcdAdCUfadTdAsAf fcAfcAfgUfusUfsu D1404 S1404 aacAgugUucuUgcuCuau AS1404 uUaUAgAGCaAGAaCACuGUUs 0.12 0.39 0.79 Asa usu D1405 S1405 AacaGuguUcuuGcucUau AS1405 uUAUaGAGcAAGaACAcUGUus 0.12 0.37 0.77 asA Usu D1406 S1406 AfaCfaAfuGfuUfcUfud AS1406 udTdAUfaGfadGdCAfaGfaG 0.59 0.93 0.89 GdCAfcUfadTdAsAf fcAfcAfgUfusUfsu D1407 S1407 aACagUGuuCUugCUcuAU AS1407 UUauAGagCAagAAcaCUguUs 0.09 0.16 0.55 asa Usu D1408 S1408 AfaCfaAfuGfuUfcUfuG AS1408 udTdAdTdAGfaGfcAfaGfaA 0.22 0.64 0.86 fcAfcdTdAdTdAsAf fcAfcAfgUfusUfsu D1409 S1409 aaCAguGUucUUgcUCuaU AS1409 uUaUAgaGCaaGAacACugUUs 0.13 0.31 0.76 Asa usu D1410 S1410 AfaCfaAfuGfuUfcUfuG AS1410 udTdAdTdAdGaGfcAfaGfaG 0.77 0.94 0.93 fcAfdCdTdAdTdAsAf fcAfcAfgUfusUfsu D1411 S1411 aacAfgugUfucuUfgcuC AS1411 uUfaUfAfgAfGfCfaAfGfAf 0.23 0.53 1.04 fuauAfsa aCfAfCfuGfUfUfsusu D1412 S1412 aacdAgugdTucudTgcud AS1412 udTadTdAgdAdGdCadAdGdA 0.30 0.64 0.90 CuaudAsa adCdAdCudGdTdTsusu D1413 S1413 AfaCfaGfuGfuUfcUfuG AS1413 UfUfaUfaGfaGfcAfaGfaAf 0.09 0.19 0.63 fcUfcUfaUfasa cAfcUfgUfusUfsu D1414 S1414 AfaCfaGfuGfUfUfcUfu AS1414 UfUfaUfaGfaGfcAfaGfaac 0.11 0.28 0.66 GfcUfcUfaUfasa AfcUfgUfusUfsu D1415 S1415 AfaCfaGfuGfuUfcUfuG AS1415 UfUfaUfaGfagcAfaGfaAfc 0.06 0.13 0.53 fCfUfcUfaUfasa AfcUfgUfusUfsu D1416 S1416 aacaguguucuugcucuau AS1416 UfUfAfUfAfGfAfGfCfAfAf 0.20 0.53 0.99 asa GfAfAfCfAfCfUfGfUfUfsu su D1417 S1417 AfaCfaGfuGfuUfcUfuG AS1417 UfUfauaGfaGfcAfaGfaAfc 0.07 0.17 0.53 fcUfcUfAfUfasa AfcUfgUfusUfsu D1418 S1418 aAfCfagUfGfuuCfUfug AS1418 UfUfauAfGfagCfAfagAfAf 0.08 0.20 0.70 CfUfcuAfUfasa caCfUfguUfsUfsu D1419 S1419 AfaCfAfGfuGfuUfcUfu AS1419 uUfaUfaGfaGfcAfaGfaAfc 0.08 0.20 0.70 AfcugUfusUfsUf

Example 3: In Vitro Silencing Activity with Various Chemical Modifications on TTR siRNA

The IC₅₀ for each modified siRNA is determined in Hep3B cells by standard reverse transfection using Lipofectamine RNAiMAX. In brief, reverse transfection is carried out by adding 5 μL of Opti-MEM to 5 μL of siRNA duplex per well into a 96-well plate along with 10 μL of Opti-MEM plus 0.5 μL of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubating at room temperature for 15-20 minutes. Following incubation, 100 μL of complete growth media without antibiotic containing 12,000-15,000 Hep3B cells is then added to each well. Cells are incubated for 24 hours at 37° C. in an atmosphere of 5% CO₂ prior to lysis and analysis of ApoB and GAPDH mRNA by bDNA (Quantigene). Seven different siRNA concentrations ranging from 10 nM to 0.6 μM are assessed for IC₅₀ determination and ApoB/GAPDH for ApoB transfected cells is normalized to cells transfected with 10 nM Luc siRNA.

Abbreviation Nucleotide(s) Af 2′-F-adenosine Cf 2′-F-cytidine Gf 2′-F-guanosine Uf 2′-F-uridine A adenosine C cytidine G guanosine U uridine a 2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine s phosphorothioate linkage

TABLE 3 ANGPTL3 modified duplex SS seq (SEQ ID NOS AS seq (SEQ ID NOS Duplex Sense 845-1025, respectively, 1026-1206, respectively, RNAimax, Hep3b ID ID in order of appearance) AD ID in order of appearance) 10 nM 0.1 nM 0.025 nM D2000 S2000 UfcAfcAfaUfuAfAfGfcUfcCfu A2000 aAfaGfaAfgGfaGfcuuAfaUfu 0.036 0.274 0.233 UfcUfuUf GfuGfasAfsc D2001 S2001 UfuAfuUfgUfuCfCfUfcUfaGfu A2001 aAfaUfaAfcUfaGfaggAfaCfa 0.044 0.278 0.247 UfaUfuUf AfuAfasAfsa D2002 S2002 GfcUfaUfgUfuAfGfAfcGfaUfg A2002 uUfuUfaCfaUfcGfucuAfaCfa 0.062 0.474 0.449 UfaAfaAf UfaGfcsAfsa D2003 S2003 GfgAfcAfuGfgUfCfUfuAfaAfg A2003 aAfaGfuCfuUfuAfagaCfcAfu 0.303 1.042 0.912 AfcUfuUf GfuCfcsCfsa D2004 S2004 CfaAfaAfaCfuCfAfAfcAfuAfu A2004 aUfcAfaAfuAfuGfuugAfgUfu 0.102 0.623 0.499 UfuGfaUf UfuUfgsAfsa D2005 S2005 AfcCfaGfuGfaAfAfUfcAfaAfg A2005 uUfcUfuCfuUfuGfauuUfcAfc 0.124 0.901 0.756 AfaGfaAf UfgGfusUfsu D2006 S2006 CfaCfaAfuUfaAfGfCfuCfcUfu A2006 aAfaAfgAfaGfgAfgcuUfaAfu 0.069 0.269 0.244 CfuUfuUf UfgUfgsAfsa D2007 S2007 CfuAfuGfuUfaGfAfCfgAfuGfu A2007 uUfuUfuAfcAfuCfgucUfaAfc 0.052 0.622 0.589 AfaAfaAf AfuAfgsCfsa D2008 S2008 UfcAfaCfaUfaUfUfUfgAfuCfa A2008 aAfgAfcUfgAfuCfaaaUfaUfg 0.133 0.798 0.785 GfuCfuUf UfuGfasGfsu D2009 S2009 AfaCfuGfaGfaAfGfAfaCfuAfc A2009 uAfuAfuGfuAfgUfucuUfcUfc 0.097 0.671 0.528 AfuAfuAf AfgUfusCfsc D2010 S2010 AfcAfaUfuAfaGfCfUfcCfuUfc A2010 aAfaAfaGfaAfgGfagcUfuAfa 0.145 0.308 0.293 UfuUfuUf UfuGfusGfsa D2011 S2011 CfuCfcAfgAfgCfCfAfaAfaUfc A2011 aUfcUfuGfaUfuUfuggCfuCfu 0.122 0.882 0.938 AfaGfaUf GfgAfgsAfsu D2012 S2012 CfgAfuGfuAfaAfAfAfuUfuUfa A2012 uUfgGfcUfaAfaAfuuuUfuAfc 0.102 0.843 0.733 GfcCfaAf AfuCfgsUfsc D2013 S2013 GfuCfuUfaAfaGfAfCfuUfuGfu A2013 uAfuGfgAfcAfaAfgucUfuUfa 1.133 1.105 1.022 CfcAfuAf AfgAfcsCfsa D2014 S2014 CfaAfcAfuAfuUfUfGfaUfcAfg A2014 aAfaGfaCfuGfaUfcaaAfuAfu 0.077 0.413 0.450 UfcUfuUf GfuUfgsAfsg D2015 S2015 AfcUfgAfgAfaGfAfAfcUfaCfa A2015 uUfaUfaUfgUfaGfuucUfuCfu 0.055 0.293 0.364 UfaUfaAf CfaGfusUfsc D2016 S2016 CfcAfgAfgCfcAfAfAfaUfcAfa A2016 aAfaUfcUfuGfaUfuuuGfgCfu 0.080 0.650 0.499 GfaUfuUf CfuGfgsAfsg D2017 S2017 GfaUfgUfaAfaAfAfUfuUfuAfg A2017 aUfuGfgCfuAfaAfauuUfuUfa 0.076 0.605 0.579 CfcAfaUf CfaUfcsGfsu D2018 S2018 UfcUfuAfaAfgAfCfUfuUfgUfc A2018 uUfaUfgGfaCfaAfaguCfuUfu 1.326 1.098 0.927 CfaUfaAf AfaGfasCfsc D2019 S2019 AfaCfaUfaUfuUfGfAfuCfaGfu A2019 aAfaAfgAfcUfgAfucaAfaUfa 0.047 0.560 0.477 CfuUfuUf UfgUfusGfsa D2020 S2020 CfuGfaGfaAfgAfAfCfuAfcAfu A2020 uUfuAfuAfuGfuAfguuCfuUfc 0.066 0.690 0.681 AfuAfaAf UfcAfgsUfsu D2021 S2021 AfaUfuAfaGfcUfCfCfuUfcUfu A2021 aUfaAfaAfaGfaAfggaGfcUfu 0.041 0.611 0.251 UfuUfaUf AfaUfusGfsu D2022 S2022 AfaAfuCfaAfgAfUfUfuGfcUfa A2022 uAfaCfaUfaGfcAfaauCfuUfg 0.053 0.555 0.516 UfgUfuAf AfuUfusUfsg D2023 S2023 UfuCfaGfuUfgGfGfAfcAfuGfg A2023 uAfaGfaCfcAfuGfuccCfaAfc 0.779 1.045 0.963 UfcUfuAf UfgAfasGfsg D2024 S2024 GfgGfcCfaAfaUfUfAfaUfgAfc A2024 aAfuAfuGfuCfaUfuaaUfuUfg 1.487 0.949 0.883 AfuAfuUf GfcCfcsUfsu D2025 S2025 AfcAfuAfuUfuGfAfUfcAfgUfc A2025 aAfaAfaGfaCfuGfaucAfaAfu 0.043 0.432 0.477 UfuUfuUf AfuGfusUfsg D2026 S2026 AfgAfaCfuAfcAfUfAfuAfaAfc A2026 uUfgUfaGfuUfuAfuauGfuAfg 0.324 1.042 0.905 UfaCfaAf UfuCfusUfsc D2027 S2027 AfuUfaAfgCfuCfCfUfuCfuUfu A2027 aAfuAfaAfaAfgAfaggAfgCfu 0.042 0.283 0.224 UfuAfuUf UfaAfusUfsg D2028 S2028 AfgAfuUfuGfcUfAfUfgUfuAfg A2028 aUfcGfuCfuAfaCfauaGfcAfa 0.349 0.936 0.896 AfcGfaUf AfuCfusUfsg D2029 S2029 UfcAfgUfuGfgGfAfCfaUfgGfu A2029 uUfaAfgAfcCfaUfgucCfcAfa 0.914 0.907 0.944 CfuUfaAf CfuGfasAfsg D2030 S2030 GfgCfcAfaAfuUfAfAfuGfaCfa A2030 aAfaUfaUfgUfcAfuuaAfuUfu 0.047 0.353 0.326 UfaUfuUf GfgCfcsCfsu D2031 S2031 CfaUfaUfuUfgAfUfCfaGfuCfu A2031 uAfaAfaAfgAfcUfgauCfaAfa 0.110 0.867 0.842 UfuUfuAf UfaUfgsUfsu D2032 S2032 UfaCfaUfaUfaAfAfCfuAfcAfa A2032 uUfgAfcUfuGfuAfguuUfaUfa 0.200 0.699 0.656 GfuCfaAf UfgUfasGfsu D2033 S2033 UfuUfuAfuUfgUfUfCfcUfcUfa A2033 aUfaAfcUfaGfaGfgaaCfaAfu 0.050 0.218 0.192 GfuUfaUf AfaAfasAfsg D2034 S2034 UfuGfcUfaUfgUfUfAfgAfcGfa A2034 uUfaCfaUfcGfuCfuaaCfaUfa 0.096 0.792 0.640 UfgUfaAf GfcAfasAfsu D2035 S2035 CfaGfuUfgGfgAfCfAfuGfgUfc A2035 uUfuAfaGfaCfcAfuguCfcCfa 0.127 0.936 0.890 UfuAfaAf AfcUfgsAfsa D2036 S2036 AfaAfuUfaAfuGfAfCfaUfaUfu A2036 uUfuGfaAfaUfaUfgucAfuUfa 0.061 0.683 0.668 UfcAfaAf AfuUfusGfsg D2037 S2037 GfaUfcAfgUfcUfUfUfuUfaUfg A2037 uAfgAfuCfaUfaAfaaaGfaCfu 0.157 1.010 0.723 AfuCfuAf GfaUfcsAfsa D2038 S2038 AfcAfuAfuAfaAfCfUfaCfaAfg A2038 uUfuGfaCfuUfgUfaguUfuAfu 0.047 0.532 0.525 UfcAfaAf AfuGfusAfsg D2039 S2039 UfuUfaUfuGfuUfCfCfuCfuAfg A2039 aAfuAfaCfuAfgAfggaAfcAfa 0.031 0.505 0.238 UfuAfuUf UfaAfasAfsa D2040 S2040 UfgCfuAfuGfuUfAfGfaCfgAfu A2040 uUfuAfcAfuCfgUfcuaAfcAfu 0.056 0.484 0.408 GfuAfaAf AfgCfasAfsa D2041 S2041 GfgGfaCfaUfgGfUfCfuUfaAfa A2041 aAfgUfcUfuUfaAfgacCfaUfg 0.570 0.999 0.994 GfaCfuUf UfcCfcsAfsa D2042 S2042 UfgAfcAfuAfuUfUfCfaAfaAfa A2042 uUfgAfgUfuUfuUfgaaAfuAfu 0.065 0.870 0.728 CfuCfaAf GfuCfasUfsu D2043 S2043 AfuCfaGfuCfuUfUfUfuAfuGfa A2043 aUfaGfaUfcAfuAfaaaAfgAfc 0.048 0.362 0.282 UfcUfaUf UfgAfusCfsa D2044 S2044 CfaUfaUfaAfaCfUfAfcAfaGfu A2044 uUfuUfgAfcUfuGfuagUfuUfa 0.314 0.904 0.937 CfaAfaAf UfaUfgsUfsa D2045 S2045 CfuUfgAfaCfuCfAfAfcUfcAfa A2045 aAfgUfuUfuGfaGfuugAfgUfu 0.060 0.295 0.251 AfaCfuUf CfaAfgsUfsg D2046 S2046 CfuAfcUfuCfaAfCfAfaAfaAfg A2046 uUfuCfaCfuUfuUfuguUfgAfa 0.052 0.570 0.599 UfgAfaAf GfuAfgsAfsa D2047 S2047 AfaGfaGfcAfaCfUfAfaCfuAfa A2047 uUfaAfgUfuAfgUfuagUfuGfc 0.028 0.369 0.381 CfuUfaAf UfcUfusCfsu D2048 S2048 AfaAfcAfaGfaUfAfAfuAfgCfa A2048 uUfuGfaUfgCfuAfuuaUfcUfu 0.039 0.227 0.204 UfcAfaAf GfuUfusUfsu D2049 S2049 GfcAfuAfgUfcAfAfAfuAfaAfa A2049 aUfuUfcUfuUfuAfuuuGfaCfu 0.032 0.437 0.422 GfaAfaUf AfuGfcsUfsg D2050 S2050 AfuAfuAfaAfcUfAfCfaAfgUfc A2050 uUfuUfuGfaCfuUfguaGfuUfu 0.297 0.946 0.850 AfaAfaAf AfuAfusGfsu D2051 S2051 GfaAfcUfcAfaCfUfCfaAfaAfc A2051 uUfcAfaGfuUfuUfgagUfuGfa 0.179 0.929 0.884 UfuGfaAf GfuUfcsAfsa D2052 S2052 UfaCfuUfcAfaCfAfAfaAfaGfu A2052 aUfuUfcAfcUfuUfuugUfuGfa 0.091 0.536 0.524 GfaAfaUf AfgUfasGfsa D2053 S2053 AfgAfgCfaAfcUfAfAfcUfaAfc A2053 aUfuAfaGfuUfaGfuuaGfuUfg 0.086 0.611 0.621 UfuAfaUf CfuCfusUfsc D2054 S2054 GfaUfaAfuAfgCfAfUfcAfaAfg A2054 aAfgGfuCfuUfuGfaugCfuAfu 0.058 0.676 0.591 AfcCfuUf UfaUfcsUfsu D2055 S2055 CfaUfaGfuCfaAfAfUfaAfaAfg A2055 uAfuUfuCfuUfuUfauuUfgAfc 0.048 0.630 0.674 AfaAfuAf UfaUfgsCfsu D2056 S2056 UfaUfaAfaCfuAfCfAfaGfuCfa A2056 aUfuUfuUfgAfcUfuguAfgUfu 0.072 0.534 0.459 AfaAfaUf UfaUfasUfsg D2057 S2057 AfaCfuCfaAfcUfCfAfaAfaCfu A2057 uUfuCfaAfgUfuUfugaGfuUfg 0.161 0.864 0.775 UfgAfaAf AfgUfusCfsa D2058 S2058 AfcUfuCfaAfcAfAfAfaAfgUfg A2058 uAfuUfuCfaCfuUfuuuGfuUfg 0.198 0.969 0.865 AfaAfuAf AfaGfusAfsg D2059 S2059 GfaGfcAfaCfuAfAfCfuAfaCfu A2059 aAfuUfaAfgUfuAfguuAfgUfu 0.031 0.253 0.210 UfaAfuUf GfcUfcsUfsu D2060 S2060 AfaCfcAfaCfaGfCfAfuAfgUfc A2060 uAfuUfuGfaCfuAfugcUfgUfu 0.035 0.561 0.569 AfaAfuAf GfgUfusUfsa D2061 S2061 AfgUfcAfaAfuAfAfAfaGfaAfa A2061 uUfcUfaUfuUfcUfuuuAfuUfu 0.057 0.668 0.386 UfaGfaAf GfaCfusAfsu D2062 S2062 AfgUfcAfaAfaAfUfGfaAfgAfg A2062 uUfuAfcCfuCfuUfcauUfuUfu 0.720 1.017 0.924 GfuAfaAf GfaCfusUfsg D2063 S2063 CfuUfgAfaAfgCfCfUfcCfuAfg A2063 uUfcUfuCfuAfgGfaggCfuUfu 0.324 1.020 0.963 AfaGfaAf CfaAfgsUfsu D2064 S2064 CfuUfcAfaCfaAfAfAfaGfuGfa A2064 aUfaUfuUfcAfcUfuuuUfgUfu 0.048 0.549 0.531 AfaUfaUf GfaAfgsUfsa D2065 S2065 CfaAfcUfaAfcUfAfAfcUfuAfa A2065 uUfgAfaUfuAfaGfuuaGfuUfa 0.046 0.739 0.649 UfuCfaAf GfuUfgsCfsu D2066 S2066 AfcCfaAfcAfgCfAfUfaGfuCfa A2066 uUfaUfuUfgAfcUfaugCfuGfu 0.076 0.840 0.777 AfaUfaAf UfgGfusUfsu D2067 S2067 GfaAfcCfcAfcAfGfAfaAfuUfu A2067 uAfgAfgAfaAfuUfucuGfuGfg 0.103 0.916 0.808 CfuCfuAf GfuUfcsUfsu D2068 S2068 GfaAfuAfuGfuCfAfCfuUfgAfa A2068 uUfgAfgUfuCfaAfgugAfcAfu 0.046 0.532 0.520 CfuCfaAf AfuUfcsUfsu D2069 S2069 UfgAfaAfgCfcUfCfCfuAfgAfa A2069 uUfuUfcUfuCfuAfggaGfgCfu 0.067 0.894 0.822 GfaAfaAf UfuCfasAfsg D2070 S2070 UfuCfaAfcAfaAfAfAfgUfgAfa A2070 aAfuAfuUfuCfaCfuuuUfuGfu 0.052 0.557 0.395 AfuAfuUf UfgAfasGfsu D2071 S2071 AfaCfuAfaCfuAfAfCfuUfaAfu A2071 uUfuGfaAfuUfaAfguuAfgUfu 0.025 0.220 0.232 UfcAfaAf AfgUfusGfsc D2072 S2072 CfcAfaCfaGfcAfUfAfgUfcAfa A2072 uUfuAfuUfuGfaCfuauGfcUfg 0.293 0.923 0.899 AfuAfaAf UfuGfgsUfsu D2073 S2073 AfaCfcCfaCfaGfAfAfaUfuUfc A2073 aUfaGfaGfaAfaUfuucUfgUfg 0.021 0.375 0.356 UfcUfaUf GfgUfusCfsu D2074 S2074 UfgUfcAfcUfuGfAfAfcUfcAfa A2074 uUfgAfgUfuGfaGfuucAfaGfu 0.052 0.402 0.513 CfuCfaAf GfaCfasUfsa D2075 S2075 GfaAfaGfcCfuCfCfUfaGfaAfg A2075 uUfuUfuCfuUfcUfaggAfgGfc 0.171 0.904 0.893 AfaAfaAf UfuUfcsAfsa D2076 S2076 AfaUfaUfuUfaGfAfAfgAfgCfa A2076 uUfaGfuUfgCfuCfuucUfaAfa 0.142 0.614 0.688 AfcUfaAf UfaUfusUfsc D2077 S2077 AfcUfaAfcUfaAfCfUfuAfaUfu A2077 uUfuUfgAfaUfuAfaguUfaGfu 0.020 0.312 0.316 CfaAfaAf UfaGfusUfsg D2078 S2078 CfaAfcAfgCfaUfAfGfuCfaAfa A2078 uUfuUfaUfuUfgAfcuaUfgCfu 0.026 0.313 0.393 UfaAfaAf GfuUfgsGfsu D2079 S2079 CfcAfcAfgAfaAfUfUfuCfuCfu A2079 aAfgAfuAfgAfgAfaauUfuCfu 0.012 0.596 0.345 AfuCfuUf GfuGfgsGfsu D2080 S2080 GfuCfaCfuUfgAfAfCfuCfaAfc A2080 uUfuGfaGfuUfgAfguuCfaAfg 0.054 0.503 0.456 UfcAfaAf UfgAfcsAfsu D2081 S2081 CfuCfcUfaGfaAfGfAfaAfaAfa A2081 uAfgAfaUfuUfuUfucuUfcUfa 0.050 0.596 0.531 UfuCfuAf GfgAfgsGfsc D2082 S2082 AfuUfuAfgAfaGfAfGfcAfaCfu A2082 uAfgUfuAfgUfuGfcucUfuCfu 0.064 0.806 0.928 AfaCfuAf AfaAfusAfsu D2083 S2083 CfuAfaCfuAfaCfUfUfaAfuUfc A2083 aUfuUfuGfaAfuUfaagUfuAfg 0.056 0.844 0.761 AfaAfaUf UfuAfgsUfsu D2084 S2084 CfaGfcAfuAfgUfCfAfaAfuAfa A2084 uUfcUfuUfuAfuUfugaCfuAfu 0.046 0.859 0.756 AfaGfaAf GfcUfgsUfsu D2085 S2085 GfaAfaUfaAfgAfAfAfuGfuAfa A2085 aUfgUfuUfuAfcAfuuuCfuUfa 0.039 0.615 0.612 AfaCfaUf UfuUfcsAfsu D2086 S2086 UfcAfcUfuGfaAfCfUfcAfaCfu A2086 uUfuUfgAfgUfuGfaguUfcAfa 0.057 0.724 0.663 CfaAfaAf GfuGfasCfsa D2087 S2087 UfcUfaCfuUfcAfAfCfaAfaAfa A2087 uUfcAfcUfuUfuUfguuGfaAfg 0.732 1.028 0.915 GfuGfaAf UfaGfasAfsu D2088 S2088 UfuUfaGfaAfgAfGfCfaAfcUfa A2088 uUfaGfuUfaGfuUfgcuCfuUfc 0.061 0.795 0.785 AfcUfaAf UfaAfasUfsa D2089 S2089 AfaAfaCfaAfgAfUfAfaUfaGfc A2089 uUfgAfuGfcUfaUfuauCfuUfg 0.330 1.017 0.865 AfuCfaAf UfuUfusUfsc D2090 S2090 AfgCfaUfaGfuCfAfAfaUfaAfa A2090 uUfuCfuUfuUfaUfuugAfcUfa 0.038 0.606 0.589 AfgAfaAf UfgCfusGfsu D2091 S2091 AfgAfcCfcAfgCfAfAfcUfcUfc A2091 aAfcUfuGfaGfaGfuugCfuGfg 0.301 0.850 0.753 AfaGfuUf GfuCfusGfsa D2092 S2092 AfgUfcCfaUfgGfAfCfaUfuAfa A2092 uUfgAfaUfuAfaUfgucCfaUfg 0.407 0.791 0.726 UfuCfaAf GfaCfusAfsc D2093 S2093 GfaUfgGfaUfcAfCfAfaAfaCfu A2093 aUfuGfaAfgUfuUfuguGfaUfc 0.120 0.658 0.654 UfcAfaUf CfaUfcsUfsa D2094 S2094 CfuAfgAfgAfaGfAfUfaUfaCfu A2094 uAfuGfgAfgUfaUfaucUfuCfu 0.071 0.610 0.645 CfcAfuAf CfuAfgsGfsc D2095 S2095 AfaAfgAfcAfaCfAfAfaCfaUfu A2095 aAfuAfuAfaUfgUfuugUfuGfu 0.029 0.306 0.461 AfuAfuUf CfuUfusCfsc D2096 S2096 CfaUfuAfuAfuUfGfAfaUfaUfu A2096 aAfaAfgAfaUfaUfucaAfuAfu 0.031 0.510 0.595 CfuUfuUf AfaUfgsUfsu D2097 S2097 GfaCfcCfaGfcAfAfCfuCfuCfa A2097 aAfaCfuUfgAfgAfguuGfcUfg 0.075 0.697 0.845 AfgUfuUf GfgUfcsUfsg D2098 S2098 GfgAfuCfaCfaAfAfAfcUfuCfa A2098 uUfcAfuUfgAfaGfuuuUfgUfg 0.130 0.831 0.951 AfuGfaAf AfuCfcsAfsu D2099 S2099 GfaAfgAfuAfuAfCfUfcCfaUfa A2099 uUfcAfcUfaUfgGfaguAfuAfu 0.058 0.828 0.938 GfuGfaAf CfuUfcsUfsc D2100 S2100 GfaCfaAfcAfaAfCfAfuUfaUfaU A2100 uUfcAfaUfaUfaAfuguUfuGfu 0.026 0.564 0.856 fuGfaAf UfgUfcsUfsu D2101 S2101 GfgGfaAfaUfcAfCfGfaAfaCfcA A2101 uAfgUfuGfgUfuUfcguGfaUfu 0.314 0.948 1.033 faCfuAf UfcCfcsAfsa D2102 S2102 AfcCfcAfgCfaAfCfUfcUfcAfaG A2102 aAfaAfcUfuGfaGfaguUfgCfu 0.033 0.448 0.675 fuUfuUf GfgGfusCfsu D2103 S2103 GfgAfcAfuUfaAfUfUfcAfaCfaU A2103 uUfcGfaUfgUfuGfaauUfaAfu 0.156 0.897 0.912 fcGfaAf GfuCfcsAfsu D2104 S2104 GfaUfcAfcAfaAfAfCfuUfcAfaU A2104 uUfuCfaUfuGfaAfguuUfuGfu 0.056 0.619 0.769 fgAfaAf GfaUfcsCfsa D2105 S2105 AfcUfcCfaUfaGfUfGfaAfgCfaA A2105 uAfgAfuUfgCfuUfcacUfaUfg 0.100 0.823 0.925 fuCfuAf GfaGfusAfsu D2106 S2106 AfcAfaCfaAfaCfAfUfuAfuAfuU A2106 aUfuCfaAfuAfuAfaugUfuUfg 0.035 0.565 0.843 fgAfaUf UfuGfusCfsu D2107 S2107 GfgAfaAfuCfaCfGfAfaAfcCfaA A2107 aUfaGfuUfgGfuUfucgUfgAfu 0.076 0.701 0.890 fcUfaUf UfuCfcsCfsa D2108 S2108 CfcCfaGfcAfaCfUfCfuCfaAfgU A2108 aAfaAfaCfuUfgAfgagUfuGfc 0.057 0.626 0.884 fuUfuUf UfgGfgsUfsc D2109 S2109 GfaCfaUfuAfaUfUfCfaAfcAfuC A2109 aUfuCfgAfuGfuUfgaaUfuAfa 0.160 0.873 1.012 fgAfaUf UfgUfcsCfsa D2110 S2110 AfaCfgUfgGfgAfGfAfaCfuAfcA A2110 uAfuUfuGfuAfgUfucuCfcCfa 0.101 0.881 0.981 faAfuAf CfgUfusUfsc D2111 S2111 CfuCfcAfuAfgUfGfAfaGfcAfaU A2111 uUfaGfaUfuGfcUfucaCfuAfu 0.026 0.435 0.691 fcUfaAf GfgAfgsUfsa D2112 S2112 CfaAfcAfaAfcAfUfUfaUfaUfuG A2112 uAfuUfcAfaUfaUfaauGfuUfu 0.154 0.882 1.091 faAfuAf GfuUfgsUfsc D2113 S2113 GfaAfaUfcAfcGfAfAfaCfcAfaC A2113 uAfuAfgUfuGfgUfuucGfuGfa 0.045 0.764 1.004 fuAfuAf UfuUfcsCfsc D2114 S2114 CfuCfuCfaAfgUfUfUfuUfcAfuG A2114 uAfgAfcAfuGfaAfaaaCfuUfg 0.105 0.925 0.988 fuCfuAf AfgAfgsUfsu D2115 S2115 AfcAfuUfaAfuUfCfAfaCfaUfcG A2115 uAfuUfcGfaUfgUfugaAfuUfa 0.114 0.919 0.905 faAfuAf AfuGfusCfsc D2116 S2116 GfgGfaGfaAfcUfAfCfaAfaUfaU A2116 aAfcCfaUfaUfuUfguaGfuUfc 0.234 1.023 0.951 fgGfuUf UfcCfcsAfsc D2117 S2117 UfcCfaUfaGfuGfAfAfgCfaAfuC A2117 aUfuAfgAfuUfgCfuucAfcUfa 0.033 0.566 0.778 fuAfaUf UfgGfasGfsu D2118 S2118 AfaCfaAfaCfaUfUfAfuAfuUfgA A2118 aUfaUfuCfaAfuAfuaaUfgUfu 0.031 0.535 0.785 faUfaUf UfgUfusGfsu D2119 S2119 UfgGfcAfaUfgUfCfCfcCfaAfuG A2119 aUfuGfcAfuUfgGfggaCfaUfu 0.065 0.815 0.967 fcAfaUf GfcCfasGfsu D2120 S2120 UfcAfgGfuAfgUfCfCfaUfgGfaC A2120 uAfaUfgUfcCfaUfggaCfuAfc 0.223 0.825 0.924 faUfuAf CfuGfasUfsa D2121 S2121 UfuAfaUfuCfaAfCfAfuCfgAfaU A2121 aUfcUfaUfuCfgAfuguUfgAfa 0.083 0.781 0.915 faGfaUf UfuAfasUfsg D2122 S2122 GfgAfgAfaCfuAfCfAfaAfuAfuG A2122 aAfaCfcAfuAfuUfuguAfgUfu 0.079 0.680 0.767 fgUfuUf CfuCfcsCfsa D2123 S2123 CfcAfuAfgUfgAfAfGfcAfaUfcU A2123 aAfuUfaGfaUfuGfcuuCfaCfu 0.026 0.537 0.793 faAfuUf AfuGfgsAfsg D2124 S2124 AfcAfaAfcAfuUfAfUfaUfuGfaA A2124 aAfuAfuUfcAfaUfauaAfuGfu 0.044 0.680 0.828 fuAfuUf UfuGfusUfsg D2125 S2125 AfaUfgCfaAfuCfCfCfgGfaAfaA A2125 uUfuGfuUfuUfcCfgggAfuUfg 0.349 0.971 1.005 fcAfaAf CfaUfusGfsg D2126 S2126 CfaGfgUfaGfuCfCfAfuGfgAfcA A2126 uUfaAfuGfuCfcAfuggAfcUfa 0.070 0.548 0.546 fuUfaAf CfcUfgsAfsu D2127 S2127 UfuCfaAfcAfuCfGfAfaUfaGfaU A2127 aUfcCfaUfcUfaUfucgAfuGfu 0.225 0.958 0.967 fgGfaUf UfgAfasUfsu D2128 S2128 GfuUfgGfgCfcUfAfGfaGfaAfgA A2128 uAfuAfuCfuUfcUfcuaGfgCfc 0.765 0.969 0.922 fuAfuAf CfaAfcsCfsa D2129 S2129 CfaUfaGfuGfaAfGfCfaAfuCfuA A2129 uAfaUfuAfgAfuUfgcuUfcAfc 0.028 0.583 0.777 faUfuAf UfaUfgsGfsa D2130 S2130 AfaCfaUfuAfuAfUfUfgAfaUfaU A2130 aAfgAfaUfaUfuCfaauAfuAfa 0.249 0.916 0.981 fuCfuUf UfgUfusUfsg D2131 S2131 GfcAfaUfcCfcGfGfAfaAfaCfaA A2131 aUfcUfuUfgUfuUfuccGfgGfa 0.435 1.002 1.019 faGfaUf UfuGfcsAfsu D2132 S2132 GfgUfaGfuCfcAfUfGfgAfcAfuU A2132 aAfuUfaAfuGfuCfcauGfgAfc 0.427 0.988 0.918 faAfuUf UfaCfcsUfsg D2133 S2133 AfuCfgAfaUfaGfAfUfgGfaUfcA A2133 uUfuGfuGfaUfcCfaucUfaUfu 0.170 0.706 0.890 fcAfaAf CfgAfusGfsu D2134 S2134 CfcUfaGfaGfaAfGfAfuAfuAfcU A2134 aUfgGfaGfuAfuAfucuUfcUfc 0.033 0.543 0.733 fcCfaUf UfaGfgsCfsc D2135 S2135 GfuUfgGfaAfgAfCfUfgGfaAfaG A2135 uUfgUfcUfuUfcCfaguCfuUfc 0.137 0.975 0.944 faCfaAf CfaAfcsUfsc D2136 S2136 AfcAfuUfaUfaUfUfGfaAfuAfuU A2136 aAfaGfaAfuAfuUfcaaUfaUfa 0.114 0.882 0.940 fcUfuUf AfuGfusUfsu D2137 S2137 CfaAfuCfcCfgGfAfAfaAfcAfaA A2137 aAfuCfuUfuGfuUfuucCfgGfg 0.155 0.755 0.686 fgAfuUf AfuUfgsCfsa D2138 S2138 CfuAfcUfuGfgGfAfUfcAfcAfaA A2138 uUfgCfuUfuGfuGfaucCfcAfa 0.196 0.825 0.658 fgCfaAf GfuAfgsAfsa D2139 S2139 AfcAfaCfcUfaAfAfUfgGfuAfaA A2139 uAfuAfuUfuAfcCfauuUfaGfg 0.133 0.704 0.671 fuAfuAf UfuGfusUfsu D2140 S2140 AfuCfcAfuCfcAfAfCfaGfaUfuC A2140 uUfcUfgAfaUfcUfguuGfgAfu 0.184 0.775 0.658 faGfaAf GfgAfusCfsa D2141 S2141 AfaCfuGfaGfgCfAfAfaUfuUfaA A2141 uCfuUfuUfaAfaUfuugCfcUfc 0.076 0.682 0.777 faAfgAf AfgUfusCfsa D2142 S2142 AfgAfgUfaUfgUfGfUfaAfaAfaU A2142 aCfaGfaUfuUfuUfacaCfaUfa 0.448 0.659 0.761 fcUfgUf CfuCfusGfsu D2143 S2143 AfaUfcCfcGfgAfAfAfaCfaAfaG A2143 aAfaUfcUfuUfgUfuuuCfcGfg 0.097 0.844 0.924 faUfuUf GfaUfusGfsc D2144 S2144 UfaCfuUfgGfgAfUfCfaCfaAfaG A2144 uUfuGfcUfuUfgUfgauCfcCfa 0.084 0.875 0.947 fcAfaAf AfgUfasGfsa D2145 S2145 CfaAfcCfuAfaAfUfGfgUfaAfaU A2145 uUfaUfaUfuUfaCfcauUfuAfg 0.104 0.811 0.814 faUfaAf GfuUfgsUfsu D2146 S2146 UfuGfaAfuGfaAfCfUfgAfgGfcA A2146 aAfuUfuGfcCfuCfaguUfcAfu 0.046 0.549 0.680 faAfuUf UfcAfasAfsg D2147 S2147 AfcUfgAfgGfcAfAfAfuUfuAfaA A2147 uCfcUfuUfuAfaAfuuuGfcCfu 0.079 0.890 1.005 faGfgAf CfaGfusUfsc D2148 S2148 GfaGfuAfuGfuGfUfAfaAfaAfuC A2148 uAfcAfgAfuUfuUfuacAfcAfu 0.497 0.676 0.783 fuGfuAf AfcUfcsUfsg D2149 S2149 AfcUfuGfgGfaUfCfAfcAfaAfgC A2149 uUfuUfgCfuUfuGfugaUfcCfc 0.049 0.699 0.907 faAfaAf AfaGfusAfsg D2150 S2150 AfuGfgUfaAfaUfAfUfaAfcAfaA A2150 uUfgGfuUfuGfuUfauaUfuUfa 0.093 0.928 0.941 fcCfaAf CfcAfusUfsu D2151 S2151 UfgAfaUfgAfaCfUfGfaGfgCfaA A2151 aAfaUfuUfgCfcUfcagUfuCfa 0.201 0.736 0.885 faUfuUf UfuCfasAfsa D2152 S2152 CfuGfaGfgCfaAfAfUfuUfaAfaA A2152 uGfcCfuUfuUfaAfauuUfgCfc 0.071 0.938 0.872 fgGfcAf UfcAfgsUfsu D2153 S2153 AfgUfaUfgUfgUfAfAfaAfaUfcU A2153 uUfaCfaGfaUfuUfuuaCfaCfa 0.504 0.816 0.689 fgUfaAf UfaCfusCfsu D2154 S2154 GfaAfaAfcAfaAfGfAfuUfuGfgU A2154 aAfaCfaCfcAfaAfucuUfuGfu 0.061 0.723 0.922 fgUfuUf UfuUfcsCfsg D2155 S2155 AfgUfgUfgGfaGfAfAfaAfcAfaC A2155 uUfaGfgUfuGfuUfuucUfcCfa 0.071 0.689 0.869 fcUfaAf CfaCfusCfsa D2156 S2156 GfuCfuCfaAfaAfUfGfgAfaGfgU A2156 uAfuAfaCfcUfuCfcauUfuUfg 0.133 0.643 0.974 fuAfuAf AfgAfcsUfsu D2157 S2157 GfaAfuGfaAfcUfGfAfgGfcAfaA A2157 uAfaAfuUfuGfcCfucaGfuUfc 0.204 0.751 1.008 fuUfuAf AfuUfcsAfsa D2158 S2158 UfgAfgGfcAfaAfUfUfuAfaAfaG A2158 uUfgCfcUfuUfuAfaauUfuGfc 0.089 0.820 0.937 fgCfaAf CfuCfasGfsu D2159 S2159 GfuAfuGfuGfuAfAfAfaAfuCfuG A2159 aUfuAfcAfgAfuUfuuuAfcAfc 0.535 0.697 0.788 fuAfaUf AfuAfcsUfsc D2160 S2160 AfaAfaCfaAfaGfAfUfuUfgGfuG A2160 aAfaAfcAfcCfaAfaucUfuUfg 0.297 0.954 1.004 fuUfuUf UfuUfusCfsc D2161 S2161 GfuGfuGfgAfgAfAfAfaCfaAfcC A2161 uUfuAfgGfuUfgUfuuuCfuCfc 0.178 0.872 0.918 fuAfaAf AfcAfcsUfsc D2162 S2162 AfuGfgAfaGfgUfUfAfuAfcUfcU A2162 uUfaUfaGfaGfuAfuaaCfcUfu 0.026 0.489 0.890 faUfaAf CfcAfusUfsu D2163 S2163 AfaUfgAfaCfuGfAfGfgCfaAfaU A2163 uUfaAfaUfuUfgCfcucAfgUfu 0.111 0.789 0.859 fuUfaAf CfaUfusCfsa D2164 S2164 GfaGfgCfaAfaUfUfUfaAfaAfgG A2164 aUfuGfcCfuUfuUfaaaUfuUfg 0.241 0.956 0.869 fcAfaUf CfcUfcsAfsg D2165 S2165 UfaUfgUfgUfaAfAfAfaUfcUfgU A2165 uAfuUfaCfaGfaUfuuuUfaCfa 0.571 0.762 0.931 faAfuAf CfaUfasCfsu D2166 S2166 AfcAfaAfgAfuUfUfGfgUfgUfuU A2166 uAfgAfaAfaCfaCfcaaAfuCfu 0.106 0.981 0.924 fuCfuAf UfuGfusUfsu D2167 S2167 UfgUfgGfaGfaAfAfAfcAfaCfcU A2167 aUfuUfaGfgUfuGfuuuUfcUfc 0.064 0.765 0.902 faAfaUf CfaCfasCfsu D2168 S2168 UfgGfaAfgGfuUfAfUfaCfuCfuA A2168 uUfuAfuAfgAfgUfauaAfcCfu 0.029 0.675 0.859 fuAfaAf UfcCfasUfsu D2169 S2169 AfuGfaAfcUfgAfGfGfcAfaAfuU A2169 uUfuAfaAfuUfuGfccuCfaGfu 0.054 0.733 0.843 fuAfaAf UfcAfusUfsc D2170 S2170 AfgGfcAfaAfuUfUfAfaAfaGfgC A2170 uAfuUfgCfcUfuUfuaaAfuUfu 0.075 0.754 0.881 faAfuAf GfcCfusCfsa D2171 S2171 AfaGfaUfuUfgGfUfGfuUfuUfcU A2171 aAfgUfaGfaAfaAfcacCfaAfa 0.303 1.065 0.977 faCfuUf UfcUfusUfsg D2172 S2172 AfaAfcAfaCfcUfAfAfaUfgGfuA A2172 uAfuUfuAfcCfaUfuuaGfgUfu 0.101 0.855 0.880 faAfuAf GfuUfusUfsc D2173 S2173 AfuAfcUfcUfaUfAfAfaAfuCfaA A2173 uUfgGfuUfgAfuUfuuaUfaGfa 0.107 0.961 0.960 fcCfaAf GfuAfusAfsa D2174 S2174 UfgAfaCfuGfaGfGfCfaAfaUfuU A2174 uUfuUfaAfaUfuUfgccUfcAfg 0.078 0.714 0.878 faAfaAf UfuCfasUfsu D2175 S2175 GfgCfaAfaUfuUfAfAfaAfgGfcA A2175 uUfaUfuGfcCfuUfuuaAfaUfu 0.054 0.767 0.918 faUfaAf UfgCfcsUfsc D2176 S2176 UfuUfuCfuAfcUfUfGfgGfaUfcA A2176 uUfuGfuGfaUfcCfcaaGfuAfg 0.915 1.030 0.916 fcAfaAf AfaAfasCfsa D2177 S2177 AfaCfaAfcCfuAfAfAfuGfgUfaA A2177 aUfaUfuUfaCfcAfuuuAfgGfu 0.042 0.260 0.448 faUfaUf UfgUfusUfsu D2178 S2178 UfaCfuCfuAfuAfAfAfaUfcAfaC A2178 uUfuGfgUfuGfaUfuuuAfuAfg 0.063 0.897 0.869 fcAfaAf AfgUfasUfsa D2179 S2179 GfaAfcUfgAfgGfCfAfaAfuUfuA A2179 uUfuUfuAfaAfuUfugcCfuCfa 0.178 0.858 0.869 faAfaAf GfuUfcsAfsu D2180 S2180 CfaGfaGfuAfuGfUfGfuAfaAfaA A2180 aAfgAfuUfuUfuAfcacAfuAfc 0.436 0.677 0.813 fuCfuUf UfcUfgsUfsg

Example 4: In Vitro Silencing Activity with Various Chemical Modifications on ANGPTL3 siRNA Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.41 of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.41 of H₂O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR 2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat #Hs00205581 ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. AD-1955 sequence, used as a negative control, targets luciferase and has the following sequence:

(SEQ ID NO: 1207) sense: cuuAcGcuGAGuAcuucGAdTsdT; (SEQ ID NO: 1208) antisense: UCGAAGuACUcAGCGuAAGdTsdT.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. 

1. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I): (I) 5′ n_(p)-N_(a)-(X X X )_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z )_(j)-N_(a)-n_(q) 3′

wherein: i and j are each independently 0 or 1; p and q are each independently 0-6; each N_(a) independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N_(b) independently represents an oligonucleotide sequence comprising 2, 3, 4, 5, or 6 2′-O-methyl modified nucleotides; each n_(p) and n_(q) independently represent an overhang nucleotide; wherein N_(b) and Y do not have the same modification; wherein XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides; and wherein the antisense strand of the dsRNA comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand, counting from the 5′ end of the antisense strand.
 2. The double-stranded RNAi agent of claim 1, wherein YYY is all 2′-F modified nucleotides.
 3. The double-stranded RNAi agent of claim 1, wherein the YYY motif occurs at or near the cleavage site of the sense strand.
 4. The double-stranded RNAi agent of claim 1, wherein the YYY motif occurs at the 9, 10 and 11 positions of the sense strand from the 5′-end.
 5. The double-stranded RNAi agent of claim 1, wherein i and j are
 0. 6. (canceled)
 7. The double-stranded RNAi agent of claim 1, wherein double-stranded RNAi agent comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).
 8. The double-stranded RNAi agent of claim 1, wherein double-stranded RNAi agent comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).
 9. The double-stranded RNAi agent of claim 1, wherein the duplex region is 17-30 nucleotide pairs in length.
 10. The double-stranded RNAi agent of claim 9, wherein the duplex region is 27-30 nucleotide pairs in length.
 11. The double-stranded RNAi agent of claim 1, wherein each strand has 17-30 nucleotides.
 12. The double-stranded RNAi agent of claim 1, wherein the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, and combinations thereof.
 13. The double-stranded RNAi agent of claim 12, wherein the nucleotides are modified with either 2′-OCH₃ or 2′-F.
 14. The double-stranded RNAi agent of claim 1, wherein the modifications on the nucleotides are selected from the group consisting of 2′-O-methyl nucleotide, 2′-deoxyfluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, 2′-ara-F, and combinations thereof.
 15. The double-stranded RNAi agent of claim 1, further comprising at least one ligand.
 16. The double-stranded RNAi agent of claim 15, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
 17. The double-stranded RNAi agent of claim 15, wherein the ligand is attached to the 3′ end of the sense strand.
 18. The double-stranded RNAi agent of claim 1, wherein the base pair at the 1 position of the 5′-end of the duplex is an AU base pair.
 19. A pharmaceutical composition comprising the double-stranded RNAi agent according to claim 1 alone or in combination with a pharmaceutically acceptable carrier or excipient.
 20. A method for inhibiting the expression of a target gene in a subject comprising the step of administering the double-stranded RNAi agent according to claim 1 to the subject, in an amount sufficient to inhibit expression of the target gene.
 21. The method of claim 20, wherein the double-stranded RNAi agent is administered through subcutaneous or intravenous administration.
 22. A method for delivering a polynucleotide to a specific target of a subject, the method comprising: delivering the double-stranded RNAi agent according to claim 1 by subcutaneous administration into the subject, such that the polynucleotide is delivered into specific target of the subject. 